WO2011150221A2 - Composés et procédés de modulation de l'efficacité bioénergétique mitochondriale par interaction avec l'atp synthase (complexe v) et ses sous-unités - Google Patents

Composés et procédés de modulation de l'efficacité bioénergétique mitochondriale par interaction avec l'atp synthase (complexe v) et ses sous-unités Download PDF

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WO2011150221A2
WO2011150221A2 PCT/US2011/038159 US2011038159W WO2011150221A2 WO 2011150221 A2 WO2011150221 A2 WO 2011150221A2 US 2011038159 W US2011038159 W US 2011038159W WO 2011150221 A2 WO2011150221 A2 WO 2011150221A2
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Prior art keywords
compound
mitochondrial
atp synthase
dexpramipexole
synthase complex
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PCT/US2011/038159
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English (en)
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WO2011150221A3 (fr
Inventor
Valentin Gribkoff
Steven Dworetzky
Elizabeth Ann Jonas
Kambiz Nassirpour Alavian
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Knopp Neurosciences, Inc.
Yale University
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Priority to US13/699,304 priority Critical patent/US20130273557A1/en
Publication of WO2011150221A2 publication Critical patent/WO2011150221A2/fr
Publication of WO2011150221A3 publication Critical patent/WO2011150221A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria

Definitions

  • Embodiments of the present invention relate to novel compounds and methods of identifying or screening compounds suitable for the treatment of diseases where mitochondrial ATP synthase plays a significant role. Embodiments of the present invention also relate to specific compounds identified by such methods, wherein the compound interacts, binds or otherwise modulates mitochondrial ATP synthase.
  • Figure 1 shows dexpramipexole inhibited PSI-induced currents in brain-derived mitochondria.
  • Figure 2 shows cyclosporine A (CSA) reduced peak conductance in dexpramipexole-sensitive PSI-mitochondria, and high calcium (Ca2+) induced dexpramipexole- sensitive currents in normal brain mitochondria, but dexpramipexole did not inhibit mitochondrial permeability transition recorded in rat liver mitochondria.
  • CSA cyclosporine A
  • Figure 3 shows dexpramipexole and CSA decreased conductance of submitochondrial vesicles (SMVs).
  • Figure 4 shows the modulation of cellular bioenergetics by dexpramipexole.
  • Figure 5 shows that dexpramipexole altered respiration parameters and ATP production in the C2C12 myoblast cell line.
  • Figure 6 shows the modulation of complex V activity by dexpramipexole; Urea- treatment of SMVs alters enzymatic activity, pharmacology of membrane currents and radiolabeled dexpramipexole binding; and binding of radiolabeled dexpramipexole to individual heterologously-expressed subunits of complex V and competition by unlabeled dexpramipexole.
  • Optical Isomers Diastereomers— Geometric Isomers— Tautomers.
  • Compounds described herein may contain an asymmetric center and may thus exist as enantiomers. Where the compounds according to the invention possess two or more asymmetric centers, they may additionally exist as diastereomers.
  • the present invention includes all such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers.
  • the formulas are shown without a definitive stereochemistry at certain positions.
  • the present invention includes all stereoisomers of such formulas and pharmaceutically acceptable salts thereof.
  • Diastereoisomeric pairs of enantiomers may be separated by, for example, fractional crystallization from a suitable solvent, and the pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid or base as a resolving agent or on a chiral HPLC column. Further, any enantiomer or diastereomer of a compound of the general formula may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
  • the term "about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
  • administering when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted.
  • administering a composition may be accomplished by oral administration, injection, infusion, absorption or by any method in combination with other known techniques.
  • animal as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.
  • inhibiting includes the administration of a compound of the present invention to prevent the onset of the symptoms, alleviating the symptoms, or eliminating the disease, condition or disorder.
  • inhibiting refers to at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% inhibition.
  • inhibiting refers to at least about 10-99, 20-99, 30-99, 40-99, 50-99, 60-99, 70-99, 80-99, or 90-99% inhibition
  • the inhibition need not be 100%. That is in the absence of a compound the disease would progress more rapidly, then as compared to in the presence of a compound, then the compound is said to inhibit the progression of the disease.
  • a compound can inhibit the progression of a disease completely or incompletely.
  • An incomplete inhibition means that the disease may progress but at a slower rate than in the absence of the compound.
  • Progression of a disease can be measured by known criteria. For example, for a neurodegenerative disease, progression can be measured/determined by measuring cognitive ability, motor activity, and the like.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • target refers to the material for which either deactivation, rupture, disruption or destruction or preservation, maintenance, restoration or improvement of function or state is desired.
  • diseased cells, pathogens, or infectious material may be considered undesirable material in a diseased subject and may be a target for therapy.
  • improves is used to convey that the present invention changes either the appearance, form, characteristics and/or physical attributes of the tissue to which it is being provided, applied or administered.
  • Improves may also refer to the overall physical state of an individual to whom an active agent has been administered. For example, the overall physical state of an individual may "improve” if one or more symptoms of a neurodegenerative disorder are alleviated by administration of an active agent.
  • terapéutica means an agent utilized to treat, combat, ameliorate or prevent an unwanted condition or disease of a patient.
  • terapéuticaally effective amount or “therapeutic dose” as used herein are interchangeable and may refer to the amount of an active agent or pharmaceutical compound or composition that elicits a biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • a biological or medicinal response may include, for example, one or more of the following: (1) preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display pathology or symptoms of the disease, condition or disorder, (2) inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptoms of the disease, condition or disorder or arresting further development of the pathology and/or symptoms of the disease, condition or disorder, and (3) ameliorating a disease, condition or disorder in an individual that is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder or reversing the pathology and/or symptoms experienced or exhibited by the individual.
  • neuroprotectant refers to any agent that may prevent, ameliorate or slow the progression of neuronal degeneration and/or neuronal cell death, or protects neurons from the toxic actions of another agent.
  • a “neuroprotectant” can also refer to any agent that may prevent, ameliorate or slow the progression of a central nervous cell degeneration and/or central nervous cell death, or protects a central nervous cell from the toxic actions of another agent.
  • central nervous system cell refers to a cell that is part of the central nervous system. Examples include, but are not limited to neurons and glia cells. Examples of neurons and glia cells include, but are not limited to, oligodendrocytes, sensory neurons, motor neurons, Schwann cells, astrocytes, and the like.
  • a “salt” is any acid addition salt, preferably a pharmaceutically acceptable acid addition salt, including but not limited to, halogenic acid salts such as hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acid salt such as, for example, nitric, perchloric, sulfuric and phosphoric acid salt; an organic acid salt such as, for example, sulfonic acid salts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or /?-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid salts; and an amino acid salt such as aspartic or glutamic acid salt.
  • halogenic acid salts such as hydrobromic, hydrochloric, hydro
  • the acid addition salt may be a mono- or di-acid addition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di- organic acid salt.
  • the acid addition salt is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this disclosure.
  • “Pharmaceutically acceptable salt” is meant to indicate those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. (1977) J. Pharm. Sciences, Vol 6. 1-19, describes pharmaceutically acceptable salts in detail.
  • treating may be taken to mean prophylaxis of a specific disorder, disease or condition, alleviation of the symptoms associated with a specific disorder, disease or condition and/or prevention of the symptoms associated with a specific disorder, disease or condition.
  • the term refers to slowing the progression of the disorder, disease or condition or alleviating the symptoms associated with the specific disorder, disease or condition.
  • the term refers to slowing the progression of the disorder, disease or condition.
  • the term refers to alleviating the symptoms associated with the specific disorder, disease or condition.
  • the term refers to restoring function which was impaired or lost due to a specific disorder, disease or condition.
  • patient generally refers to any living organism to which compounds described herein are administered and may include, but is not limited to, any non-human mammal, primate or human. Such “patients” may or my not be exhibiting the signs, symptoms or pathology of the particular diseased state.
  • KNS-760704 is dexpramipexole (((6R)-2-amino-4,5,6,7- tetrahydro-6-(propylamino)benzothiazole)), which is a synthetic aminobenzothiazole derivative.
  • the (6S) enantiomer of KNS-76704 is a potent dopamine agonist, which mimics the effects of the neurotransmitter dopamine.
  • Pramipexole has also been shown to have both neuroprotective and dopaminergic activities, presumably through inhibition of lipid peroxidation, normalization of mitochondrial metabolism and/or detoxification of oxygen radicals. Therefore, pramipexole may have utility as an inhibitor of the cell death cascades and loss of cell viability observed in neurodegenerative diseases such as Parkinson's disease. Additionally, oxidative stress caused by an increase in oxygen and other free radicals has been associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder involving the motor neurons of the cortex, brain stem, and spinal cord.
  • ALS amyotrophic lateral sclerosis
  • dexpramipexole has been demonstrated to have effects in normal and stressed mitochondria that are consistent with the interpretation that the compound increases bioenergetic efficiency by suppressing aberrant large-conductance currents in mitochondrial membranes. Such currents provide a source of uncoupling between oxygen and metabolic substrate use and oxidative phosphorylation, and may represent a leak which reduces the proton-motive force driving ATP production.
  • Dexpramipexole has been shown to be neuroprotective in multiple in vitro and in vivo assays. While its mechanism of action remains to be fully characterized, recent studies have localized the target responsible for its neuroprotective properties to the level of the mitochondrion.
  • Dexpramipexole produces a concentration-dependent inhibition of large-conductance ion channel activity produced in isolated rat brain mitochondria associated with neuronal cell death. Consistent with this pharmacology, dexpramipexole also produced significant concentration- dependent in vitro cytoprotection in human neuroblastoma SH-SY5Y cells exposed to the mitochondrial toxin MPP + or the proteasome inhibitor PSI. An independent laboratory has shown that dexpramipexole significantly increased in vivo survival in the G93A-SOD1 mutant mouse model of ALS.
  • proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leu-H is a synthetic peptide that reversibly inhibits the chymotrypsin-like activity of the proteasome and has been used in vivo to model the effects of PD and in vitro to elucidate potential neurotoxic mechanisms involved in these disorders.
  • Mitochondria derived ex vivo from subcortical brain regions of rats treated with PSI exhibit unusual large-conductance ion channel activity similar to mitochondrial transition pore activity of the inner mitochondrial membrane.
  • dexpramipexole when applied to these mitochondria under whole-cell mitochondrial voltage clamp, produces a concentration-dependent, potent, effective and reversible suppression of this conductance with little effect on lower-amplitude conductances seen in control mitochondria. Accordingly, in some embodiments, the methods described herein can be used to identify a compound that inhibits PSI's effect(s) on a cell.
  • a method that identifies a compound that can suppress the effect of PSI.
  • the method comprises contacting a cell, mitochondira, or sub-mitochondrial vesicle with PSI in the absence or presence of a test compound.
  • a test compound As described herein and is applicable to all cells, mitochondria, or sub-mitochondrial vesicles, the cells, mitochondria, or sub-mitochondrial vesicles can be isolated. The cells, mitochondria, or sub-mitochondrial vesicles can also be part of a subject or organism.
  • the effect of the test compound is measured and if the test compound suppresses the effects of PSI, the compound is said to be able to suppress the effect of PSI.
  • the test compound is compared to a positive control's ability to suppress the effect of PSI.
  • the positive control is dexpramipexole.
  • a method of identifying a compound can comprise contacting a test compound with an ATP synthase complex.
  • an ⁇ synthasome' can be used as the ATP synthase complex.
  • An ATP synthasome in some embodiments, is a preparation that isolates specific and critical components of the bioenergetic pathway of the mitochondrion, ATP synthase (complex V) and associated proteins inserted into a membrane and comprising a submitochondrial particle, and in which application of voltage through a patch clamp electrode can produce a large-conductance leak current, similar to the current described above in neuronal mitochondria derived from PSI-treated rats.
  • the ATP synthasome leak current can be inhibited by a compound identified herein.
  • the compound is dexpramipexole.
  • the compound has an EC50 of about 100-120, 110- 120, nM and any number in the range including the endpoints.
  • the concentration-response relationships for current inhibition by dexpramipexole or a compound identified by a method described herein in both mitochondria and synthasomes can be very shallow (Hill slopes «1).
  • dexpramipexole or a compound identified by a method described herein may work by decreasing an insult-dependent or stress-augmented proton (H + ) leak conductance that shunts the proton-motive force necessary to couple the electron transport chain to ATP production, and that components of the mitochondrial transition pore are localized in the complex producing the leak conductance. Attenuating, but more likely entirely inhibiting this leak conductance should improve mitochondrial bioenergetics by increasing the efficiency of ATP production. In some embodiments, such a mechanism would enable neurons to more effectively resist environmental stresses, including toxicity from abnormal protein aggregation and reactive oxygen species, which are associated with the development of many neurodegenerative diseases.
  • H + insult-dependent or stress-augmented proton
  • a compound identified by a method described herein can be used to increase mitochondrial bioenergetics.
  • the increase or improvement of mitochondrial bioenergetics comprises an increase in the efficiency of ATP production.
  • a compound identified using a method described herein can be used to inhibit leak conductance.
  • the method comprises contacting a cell or a subject with a compound identified by a method described herein, wherein the compound inhibits leak conductance.
  • dexpramipexole binds to a site in the ATP synthase complex, inhibiting a leak conductance in a cooperative manner. Therefore, additional compounds can be identified by identifying compounds that bind to the Fl head. In some embodiments, a compound that binds to the Fl head and can competitively inhibit the binding of dexpramipexole is identified as a compound that can increase mitochondrial bioenergetic efficiency. In some embodiments, the method comprises contacting a Fl head with a test compound and determining whether the test compound binds to the Fl head.
  • the method comprises contacting a Fl head with dexpramipexole (labeled or unlabeled) and a test compound either simultaneously or sequentially in any order and determining whether the test compound can bind to the Fl head or can inhibit the binding of dexpramipexole to the Fl head.
  • the test compound if the test compound can bind to the Fl head and can inhibit the binding of dexpramipexole to the Fl head, the compound is identified as a compound that can increase bioenergetic efficiency or can inhibit leak conductance.
  • an ATP synthase complex such as, but not limited to, an ATP synthasome comprises the Fl head.
  • the ATP synthase complex is an ATP synthase complex as described herein.
  • a high-throughput respirometry system can be used to show that, for example, dexpramipexole can reduce oxygen consumption by mitochondria without forcing cells to 'switch' to glycolysis to obtain needed ATP.
  • This high-throughput system can also be used to identify compounds other than dexpramipexole that can reduce oxygen consumption without an increase in glycolysis to increase ATP.
  • ATP levels in the presence of a compound, such as dexpramipexole can remain constant or are even increased over a wide range of concentrations, direct evidence that the compound can improve bioenergetic efficiency even in non-stressed cells.
  • dexpramipexole interacts (e.g. binds) with specific subunits of an ATP synthase complex.
  • nine specific subunits of ATP synthase can be heterologously expressed in a cell, such as, but not limited to, 293T cells, including the proteins comprising the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the human ORF constructs for these subunits can be tagged with Myc and DDK (Flag) tags, which can be obtained from Origene Technologies (Rockville, MD).
  • the cells can be transfected with the above constructs, using any transfection method, including a lipid mediated (e.g. LIPOFECTAMINE) or a calcium phosphate method.
  • a lipid mediated e.g. LIPOFECTAMINE
  • the cells on day 1 , 2, or 3 post-transfection, the cells can be lysed and the fusion proteins were bound to an EZviewTM Red ANTI-FLAG® M2 Affinity Gel, using a standard protocol.
  • the proteins can be eluted from a portion of the samples and presence of the proteins on the beads can be verified by immunoblot analysis, using commercially available mouse anti-Myc antibodies.
  • the protein-bound beads can then be incubated with a test compound that is labeled or unlabelled or a positive control such as dexpramipexole to determine whether the test compound can bind to the ATP synthase complex or a portion thereof.
  • a test compound that is labeled or unlabelled or a positive control such as dexpramipexole
  • a positive control such as dexpramipexole
  • Any method can be used to detect the interaction. For example, if the compound is labeled (e.g. 14 C-labeling) the amount of R elabeled compound bound to the beads can be measured to determine if the compound binds to the ATP synthase complex or a portion thereof, such as a subunit of the ATP synthase complex.
  • the same type of method can be used to determine if a compound binds to the Fl head. Examples of methods are also described in the example section.
  • the test compound can be compared to a positive control, such as dexpramipexole, or the ability for a test compound to bind to the ATP synthase complex can be measured by determining whether the test compound can inhibit the binding of the positive control to the ATP synthase complex.
  • a 14 C-labeled dexpramipexole is used as a positive control (e.g. binding ligand).
  • a method can be designed to determine whether the binding of 14 C-dexpramipexole is specific and competitive.
  • the protein- bound beads can be co-incubated in 14 C-dexpramipexole (e.g. 1 -200 nM) and non-radiolabeled dexpramipexole at a concentration of, for example, 100 ⁇ , a concentration, which can be, -500X the concentration of the radiolabeled compound.
  • 14 C-dexpramipexole e.g. 1 -200 nM
  • non-radiolabeled dexpramipexole at a concentration of, for example, 100 ⁇ , a concentration, which can be, -500X the concentration of the radiolabeled compound.
  • there can be no effect on the untransfected control level of radioactivity but the levels bound to both the b subunit and the OSCP subunit can be reduced to levels insignificantly different from the control levels.
  • dexpramipexole binds to one or more sites in the b and OSCP subunits of the FIFo ATP synthase (complex V). In some embodiments, the b and OSCP subunits are closely associated, and form the 'stator' stalk believed to have a role in stabilizing Fl during rotation. In some embodiments, dexpramipexole binds to one or more sites in the FI Fo ATP synthase 'stator' stalk. In some embodiments, the interaction with this/these sites inhibits stress- or dysfunction-induced metabolic leak conductance in mammalian mitochondria, including dysfunctional mitochondria in humans. In some embodiments, a test compound is also used to determine whether the binding is specific and competitive. The binding method can be done in the presence or absence of a positive control (e.g. dexpramipexole).
  • a positive control e.g. dexpramipexole
  • the inhibition of mitochondrial leak conductance increases bioenergetic efficiency.
  • multi-well and high-resolution respirometry systems are used with both whole cells in culture and isolated mitochondria from neurons to show the increase in bioenergetic efficiency.
  • dexpramipexole or a compound identified using a method described herein can be shown to decrease the basal consumption of oxygen (0 2 ) without, in whole cells, producing a compensatory switch in cell energy production to glycolysis.
  • the methods described herein can be used to identify a compound that decreases the basal consumption of oxygen (0 2 ) without, in whole cells, producing a compensatory switch in cell energy production to glycolysis.
  • this can be seen as shown in examples described herein, which show that in the presence of dexpramipexole, oxygen consumption rate (OCR) was decreased modestly with no accompanying change in ECAR (extracellular acidification rate), a measure of the production of lactic acid by glycolysis.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • oxygen consumption can be decreased significantly, in this case reflecting mitochondrial dysfunction, since ECAR is significantly increased. Accordingly, a compound that decreases oxygen consumption can be identified in the presence of PSI or in some other embodiments, in the absence of PSI.
  • a method of identifying a compound that decreases oxygen consumption comprises contacting a cell with a test compound in the presence or absence of PSI and measuring oxygen consumption.
  • the test compound is compared to a positive control, such as dexpramipexole, wherein when the test compound decreases oxygen consumption the same or greater than the positive control the compound is said to be a compound that decreases oxygen consumption or the rate thereof.
  • a positive control such as dexpramipexole
  • the present invention provides a method of identifying a compound that increases mitochondrial bioenergetic efficiency.
  • the method comprises contacting a test compound with a cell and measuring mitochondrial bioenergetic efficiency.
  • measuring mitochondrial bioenergetic efficiency comprises measuring oxygen consumption or the rate thereof. If the rate of oxygen consumption or the amount of oxygen consumed is decreased without a concomitant increase in glycolysis (e.g. ECAR) the compound is said to be a compound that increases mitochondrial bioenergetic efficiency.
  • the cell is also contacted with PSI, which does not increase mitochondrial bioenergetic efficiency. If the test compound can inhibit the effects of PSI, the compound is said to increase mitochondrial bioenergetic efficiency.
  • the test compound is compared to a positive control can increase mitochondrial bioenergetic efficiency.
  • a positive control is dexpramipexole.
  • a compound identified as described herein can increase maximal respiratory capacity, as well as spare respiratory capacity, a phenomenon that is particularly visible and pronounced when succinate is used as the electron donor via complex 2 of the electron transport system in the presence of the complex 1 inhibitor rotenone.
  • dexpramipexole or a compound identified using the methods described herein increases cell survival.
  • the cell survival is increased in a model of PSI-induced proteasome dysfunction where another compound such as riluzole does not.
  • the compound, such as dexpramipexole or a compound identified using a method described herein can exert a direct cytoprotective effect (e.g., not glial cell mediated) on neuronal cells challenged with proteasome inhibition under diverse treatment paradigms, and suggests that inhibition of mitochondrial 'leak' conductances likely mediate this protective effect.
  • a compound such as dexpramipexole or another identified by a method described herein or by specific interaction with one or more sites in the FIFo ATP synthase can inhibit aberrant metabolic leak conductances in mitochondria, increasing bioenergetic efficiency in these mitochondria and acting to protect cells from a variety of toxins and disease conditions.
  • the method comprises contacting a cell or administering to a subject a therapeutically effective amount of a composition comprising a compound identified using a method described herein, wherein the compound increases cell survival.
  • the composition does not comprise dexpramipexole.
  • the method of increasing cell survival is combined with any method of identifying a compound as described herein.
  • the methods described herein can be used in methods to identify other compounds that can increase cell survival, increase oxygen utilization efficiency, decrease the rate of oxygen consumption, and at least maintain ATP synthesis using the methods described herein (e.g. no increase in glycolysis).
  • the methods described herein may recite a particular readout, each of the methods described herein can also be used to identify a compound that increases cell survival.
  • the method comprises measuring cell survival in the absence or presence of a compound or as compared to a positive or negative control.
  • a positive control is a compound that increases cell survival as compared to the cell survival in the absence of the positive control.
  • the cell survival can be measured in the presence or absence of a cellular stress, such as, but not limited to, those described herein.
  • a negative control is a compound that has no effect on cell survival.
  • Some embodiments of the present invention relate to novel compounds and methods of identifying or screening compounds suitable for the treatment of diseases where mitochondrial ATP synthase plays a significant role.
  • Embodiments of the present invention also relates to specific compounds identified by such methods which interact or modulate mitochondrial ATP synthase.
  • the compounds inhibit mitochondrial ATP synthase activity.
  • ATP synthase complex may be interchanged with ATP synthase or a portion thereof. That is, the term "ATP synthase complex" can refer to the complete ATP synthase complex or a portion of the complex. In some embodiments, a portion of the ATP synthase complex is the Fl head.
  • a portion of the ATP synthase complex refers to at least one the subunits, which include, for example, alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin- sensitivity conferring protein).
  • a portion of the ATP synthase complex refers to the Fl head, the b subunit, the OSCP subunit, or any combination thereof.
  • the ATP synthase complex can also be an ATP synthasome, which is described herein.
  • the present invention provides compositions comprising a crystal of a mitochondrial ATP synthase complex, wherein the complex includes the Fl 'head' of the mitochondrial ATP synthase complex.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • the ATP synthase complex is a mitochondrial ATP synthase complex.
  • the mitochondrial ATP synthase is an isolated mitochondrial ATP synthase complex.
  • isolated mitochondrial ATP synthase complex refers to an ATP synthase complex that has been isolated (e.g. purified) away from a mitochondria.
  • the isolated ATP synthase complex can be functional or nonfunctional. Even if the ATP synthase complex is non-functional, in some embodiments, the ATP synthase complex retains its three-dimensional structure as if it were present in a mitochondria.
  • the ATP synthase complex is denatured. In some embodiments, the ATP synthase complex is not denatured.
  • assays for screening compounds that modulate the activity of mitochondrial ATP synthase are provided.
  • compounds are identified as inhibitors of mitochondrial ATP synthase via comparison to, e.g., dexpramipexole.
  • these assays may be cell-based, consisting of establishing aberrant bioenergetic respiratory profiles following induction of dysfunction and testing the ability of compounds to re-establish, induce, or enhance bioenergetic efficiency in these cells, as indicated by, for example, recording the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in response to additions of various substrates and under various experimental conditions.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • Substrates include but are not limited to succinate, glutamate, pyruvate, maleate and glucose.
  • Experimental conditions include but are not limited to addition of adenosine diphosphate (ADP) and the uncoupling compound FCCP, as well as the complex V inhibitor oligomycin.
  • ADP adenosine diphosphate
  • FCCP complex V inhibitor oligomycin
  • Cells useful in such assays may include clonal cell lines, derived from human and animal sources, as well as embryonic neurons grown in culture and other cells from animal sources.
  • Such techniques of respiratory profiling can also be used as an assay employing isolated mitochondria, which may be neuronal in origin, derived acutely from experimental animals including transgenic models of disease, or they may be derived from other organ systems, such as, but not limited to, mitochondria from liver cells or fibroblasts.
  • peripheral cells can be harvested from patients; these may be used as acutely cultured cells, or mitochondria may be derived for assays.
  • test compounds may also be based on the binding affinity of radiolabeled dexpramipexole to either native complex V in isolated mitochondrial membranes, or may utilize the heterologous expression of the b and OSCP subunits bound to beads as an assay.
  • test compounds would be used to displace radiolabeled dexpramipexole; compounds would be ranked based on the IC50 and KD of such interactions.
  • the present invention provides methods of identifying a compound that increases oxygen utilization efficiency.
  • the method comprises contacting a compound that binds a mitochondrial ATP synthase complex with a mitochondria or a sub-mitochondrial vesicle; and measuring oxygen utilization efficiency, wherein when the measured oxygen utilization efficiency in the mitochondria or a sub- mitochondrial vesicle increases in the presence of the compound that binds the mitochondrial ATP synthase complex indicates that the compound is a compound that increases oxygen utilization efficiency.
  • the increase in oxygen utilization efficiency can be determined using any method.
  • the oxygen utilization efficiency is determined by comparing the oxygen consumption rate (OCR) in the presence and absence of the compound.
  • a compound that decreases the amount of oxygen utilized by the cell while maintaining the cell's viability is a compound that increases the oxygen utilization efficiency.
  • the oxygen utilization efficiency refers to the amount of oxygen that a cell requires for growth and/or survival. Therefore, if a cell requires more oxygen in the presence of a compound the oxygen utilization efficiency is said to decrease. If a cell requires less oxygen in the presence of a compound, the oxygen utilization efficiency is said to increase.
  • the oxygen utilization efficiency can be determined by measuring and/or comparing the oxygen consumption rate as described herein and by other methods known.
  • the compounds ability to have an effect on oxygen utilization efficiency is determined by comparing the compound to a positive or a negative control.
  • a positive control compound is a compound that increases oxygen utilization efficiency.
  • a negative control compound is a compound that has no effect on oxygen utilization efficiency.
  • the compound is compared to a compound that is known to decrease oxygen utilization efficiency.
  • the positive control compound is dexpramipexole.
  • the method comprises identifying a compound that binds to the mitochondrial ATP synthase complex. In some embodiments, the method comprises contacting a test compound with the mitochondrial ATP synthase complex and identifying the test compound as the compound that binds the mitochondrial ATP synthase complex.
  • Methods of determining whether a compound can bind to another protein or protein complex are known to one of skill in the art. These methods can be adopted to determine the binding of a test compound to another protein or protein complex, such as the ATP synthase complex.
  • the mitochondria used in a method described herein are isolated mitochondria.
  • An "isolated mitochondria” is a mitochondria that is has been isolated or purified from a cell. The mitochondria can be isolated to a 100% homogeneous composition where no other organelles are present. In some embodiments, the isolated mitochondria are not 100% homogeneous, but the mitochondria have been separated from the cell or are not encompassed by cell membrane or a lipid bilayer.
  • a cell comprises the mitochondria. In some embodiments, a cell comprises the mitochondrial ATP synthase complex. In some embodiments, an isolated mitochondria comprises the mitochondrial ATP synthase complex.
  • the mitochondria used in methods described herein can be mitochondria isolated from a muscle cell or a central nervous system cell.
  • the term "cell" refers to a muscle cell or a central nervous system cell.
  • a cell is a mammalian cell.
  • the cell is a human cell.
  • a cell is a cell that is or has been isolated from a diseased central nervous system (e.g. neurological) tissue or cell population.
  • the cell is an Alzheimer's central nervous system cell, a Parkinson's central nervous system cell, an Amyotrophic lateral sclerosis cell, a Huntingdon's disease cell, and the like.
  • a cell When a cell is referred to as being a certain disease or condition central nervous system cell it is to be understood that the cell is derived from or has been isolated from a patient or subject with the disease.
  • the cell need not be a primary cell line.
  • the cell is a transformed cell.
  • the cell is not a transformed cell.
  • the cell is not actively dividing or is not capable of further cell division.
  • the cell is only capable of cell division if exposed to a mitogen (e.g. growth factor).
  • a mitogen e.g. growth factor
  • the ATP synthase complexes described herein can also be heterologously expressed.
  • the subunits necessary to form an ATP synthase complex are expressed in a cell or cell free system and the proteins are purified or isolated under conditions allowing the ATP synthase complex to form.
  • the subunits can be expressed using routine recombinant techniques such as plasmids, vectors (e.g. viral or non-viral) and the like.
  • a method that identifies a compound that increases oxygen utilization efficiency and at least maintains ATP synthesis (e.g. production).
  • the method comprises identifying a compound that increases oxygen utilization efficiency as described herein.
  • the method comprises measuring ATP synthesis in the presence of the compound, wherein when the measured ATP synthesis is at least maintained in the presence of the compound indicates that the compound at least maintains ATP synthesis.
  • a compound that increases oxygen utilization efficiency and at least maintains ATP synthesis is identified as a compound that increases oxygen utilization efficiency and at least maintains ATP synthesis. Measuring ATP synthesis can be by any method including, but not limited to, the methods described herein.
  • ATP synthesis can be measured using a luciferin-luciferase assay.
  • the luciferin-luciferase assay measures ATP hydrolysis and can be correlated with the amount of ATP synthesis.
  • a non- limiting example of this assay is described herein, but the conditions can be routinely modified to measure ATP synthesis and ATP.
  • ATP synthesis can be also be measured using an NADH assay.
  • NADH assay for example, ATP hydrolysis can be measured using an NADH- ATP-synthase kit (Mitosciences, USA; catalog # MS541).
  • the protocol can be modified according to the conditions. A non-limiting example of such a modification is described herein.
  • the compound binds to an ATP synthase complex or is identified as binding to the ATP synthase complex.
  • the compounds binds to a Fl head, a b subunit, a OSCP subunit, or a combination thereof.
  • identifying a compound as a compound that binds the mitochondrial ATP synthase complex comprises determining whether the test compound can competitively inhibit the binding of a compound known to bind, or suspected of binding, to an ATP synthase complex.
  • the compound known to bind, or suspected of binding to an ATP synthase complex is dexpramipexole.
  • the test compound or the compound known to, or suspected of binding, to an ATP synthase compound can have a detectable label.
  • the detectable label can be used to determine whether the test compound can competitively inhibit the binding of another compound to an ATP synthase complex or whether the compound itself can bind to the ATP synthase complex even in the absence of the known compound.
  • the label's signal can change when the compound binds to an ATP synthase complex.
  • the label is not detectable until the compound binds to the ATP synthase complex.
  • the compound known to, or suspected of binding to an ATP synthase complex comprises a detectable label and the ability to detect the label is abrogated or diminished when the test compound is contacted with the ATP synthase complex.
  • Other methods of detection can also be used.
  • the detectable label is a radioactive or fluorescent label.
  • the effect of the test compound on ATP synthesis can be compared to a positive or a negative control.
  • a compound is said to be a positive control if it is known to at least maintain ATP synthesis.
  • the positive control increases ATP synthesis.
  • a compound is said to be a negative control if it decreases ATP synthesis.
  • the positive control is dexpramipexole.
  • the method comprises identifying a compound that enhances ATP synthesis greater than the positive control.
  • the identified compound enhances ATP synthesis more than dexpramipexole.
  • a compound that increases ATP synthesis greater than a positive control is a compound that significantly increases ATP synthesis.
  • measuring oxygen utilization efficiency comprises measuring inhibition of mitochondrial conductance.
  • measuring oxygen utilization efficiency comprises measuring inhibition of mitochondrial conductance, which comprises measuring metabolic leak conductance.
  • measuring inhibition of mitochondrial conductance comprises a manual patch clamp recording and analysis.
  • measuring inhibition of mitochondrial conductance comprises automated patch clamp recording and analysis.
  • the automated patch clamp recording and analysis comprises a planar chip recording technique, which can also be referred to as planar patch clamp.
  • the planar patch clamp technique is described, for example in Py et al (Biotechnology and Bioengineering, Volume 107, Issue 4, pages 593-600, 1 November 2010), which is hereby incorporated by reference in its entirety.
  • the planar patch clamp technique can be used, for example, to create a high throughput screen, so that many compounds can be tested and analyzed for their ability to increase oxygen utilization efficiency and/or increase ATP synthesis.
  • the methods described herein comprise identifying a compound as a compound that treats a neurodegenerative disease.
  • the method comprises contacting a subject with a neurodegenerative disease with a compound identified that increases oxygen utilization efficiency, wherein a compound that improves the condition of the subject, inhibits the progression of the neurodegenerative disease, ameliorates the neurodegenerative disease indicates the compound as a compound that treats a neurodegenerative disease.
  • the method comprises testing a compound that has been identified as a compound that increases oxygen utilization efficiency and/or ATP synthesis using, for example, the methods described herein.
  • a subject with a neurodegenerative disease that is described herein can be, for example, a mammal, a human, a non-human primate, a dog, a cat, a monkey, a mouse, a rat, a rodent, and the like.
  • the neurodegenerative disease can be any including, but not limited to, those described herein.
  • the methods described herein comprise identifying a compound as a compound that is a neuroprotectant.
  • the method comprises contacting a subject with a neurodegenerative disease with a compound that that increases oxygen utilization efficiency, wherein a compound that improves the condition of the subject inhibits the progression of the neurodegenerative disease, ameliorates the neurodegenerative disease indicates the compound as a compound that treats a neurodegenerative disease.
  • the method comprises testing a compound that has been identified as a compound that increases oxygen utilization efficiency and/or ATP synthesis using, for example, the methods described herein.
  • the present invention provides methods of identifying a compound that increases oxygen utilization efficiency and at least maintains ATP synthesis.
  • the method comprises contacting a mitochondria or a sub-mitochondrial vesicle with a compound that binds to a mitochondrial ATP synthase complex and measuring oxygen utilization efficiency and ATP synthesis, wherein a compound that increases oxygen utilization efficiency and enhances or increases ATP synthesis identifies the compound as a compound that that increases oxygen utilization efficiency and at least maintains ATP synthesis.
  • the method comprises determining whether the compound can inhibit the binding of dexpramipexole to the mitochondrial ATP synthase complex, wherein a compound that inhibits the binding of a positive control (e.g. dexpramipexole) to the mitochondrial ATP synthase complex identifies the compound as a compound that binds to the mitochondrial synthase complex.
  • a positive control e.g. dexpramipexole
  • the method comprises comparing oxygen utilization efficiency and ATP synthesis in the presence of the compound to oxygen utilization efficiency and ATP synthesis in the presence of a positive control (e.g.
  • dexpramipexole wherein a compound that at least maintains oxygen utilization efficiency and at least maintains ATP synthesis as compared to a positive control (e.g. dexpramipexole) identifies the test compound as a compound that increases oxygen utilization efficiency and at least maintains ATP synthesis.
  • a positive control e.g. dexpramipexole
  • the positive or negative controls described herein can have a detectable label as described herein or with another detectable label.
  • the specific detectable label is not significant and any label that can be detected either upon binding or inhibition of binding can be used.
  • the methods provided by the present invention comprise identifying a compound as a compound that treats a neurodegenerative disease comprising contacting a subject with a neurodegenerative disease with a compound identified as a compound that that at least increases oxygen utilization efficiency and at least maintains ATP synthesis, wherein a compound that improves the condition of the subject; inhibits the progression of the neurodegenerative disease, or ameliorates the neurodegenerative disease indicates the compound as a compound that treats a neurodegenerative disease.
  • the present invention provides a method of identifying a compound that treats a neurodegenerative disease.
  • the method comprises contacting a subject with a neurodegenerative disease with a test compound, wherein said test compound is a compound that increases oxygen utilization efficiency, a compound that at least maintains ATP synthesis, and/or a compound that binds to a mitochondrial ATP synthase complex, wherein a compound that improves the condition of the subject; inhibits the progression of the neurodegenerative disease, ameliorates the neurodegenerative disease indicates the compound as a compound that treats a neurodegenerative disease.
  • the method comprises identifying a compound that increases oxygen utilization efficiency, a compound that at least maintains ATP synthesis, and/or a compound that binds to a mitochondrial ATP synthase complex.
  • the test compound is a compound that increases oxygen utilization efficiency, at least maintains ATP synthesis, and binds to a mitochondrial ATP synthase complex.
  • the present invention also provides, in some embodiments, a method of treating a neurodegenerative disease in a subject comprising administering to the subject a compound identified by any of the methods described herein or a combination of any of the methods described herein. Additionally, any of the methods described herein can be combined in whole or in part with each method to produce a method of identifying a compound that increases oxygen utilization efficiency, at least maintains ATP synthesis, binds to an ATP synthesis complex, or any combination thereof.
  • test compound can be tested in the methods described herein.
  • the test compound or a compound used in a method described herein is not dexpramipexole.
  • a method of identifying a neuroprotectant comprises contacting a compound that binds a mitochondrial ATP synthase complex with a mitochondria or a sub-mitochondrial vesicle; and measuring oxygen utilization efficiency, wherein when the measured oxygen utilization efficiency in the mitochondria or a sub-mitochondrial vesicle increases in the presence of the compound that binds the mitochondrial ATP synthase complex indicates that the compound is a neuroprotectant.
  • the method comprises identifying a compound that binds to the mitochondrial ATP synthase complex.
  • the method comprises contacting a test compound with the mitochondrial ATP synthase complex ; and identifying the test compound as the compound that binds the mitochondrial ATP synthase complex. In some embodiments, the method comprises measuring ATP synthesis in the presence of the compound, wherein when the measured ATP synthesis is at least maintained in the presence of the test compound which indicates that the compound is a neuroprotectant.
  • the present invention provides a method of identifying a neuroprotectant.
  • the method comprises contacting a subject with a neurodegenerative disease with a test compound, wherein the test compound is a compound that increases oxygen utilization efficiency, a compound that at least maintains ATP synthesis, and/or a compound that binds to a mitochondrial ATP synthase complex, wherein a compound that improves the condition of the subject; inhibits the progression of the neurodegenerative disease, or ameliorates the neurodegenerative disease indicates the compound as a compound that treats a neurodegenerative disease.
  • the present invention provides a method of identifying a compound that does not increase oxygen utilization efficiency comprising contacting a compound that binds a mitochondrial ATP synthase complex with a mitochondria or a sub- mitochondrial vesicle; and measuring oxygen utilization efficiency, wherein when the measured oxygen utilization efficiency in the mitochondria or a sub-mitochondrial vesicle is not increased in the presence of the compound that binds the mitochondrial ATP synthase complex indicates that the compound is a compound that does not increase oxygen utilization efficiency.
  • the method comprises measuring ATP synthesis in the presence of said the compound, wherein when the measured ATP synthesis is not maintained in the presence of the test compound indicates that the compound does not increase oxygen utilization efficiency and maintain ATP synthesis.
  • a method for identifying a compound that increases cell survival.
  • the method comprises contacting a cell with a compound that binds to an ATP synthase complex and increases oxygen utilization efficiency and measuring cell survival, wherein an increase in cell survival in the presence of the compound identifies the compound as a compound that increases cell survival.
  • the method comprises comparing the compound to a positive and/or a negative control.
  • the method comprises comparing the cell survival in the presence and the absence of the compound (e.g. test compound).
  • the present invention provides methods for preparing a mitochondrial ATP synthase complex modulating compound comprising applying a three- dimensional molecular modeling algorithm to the atomic coordinates of at least a portion of the ATP synthase complex, particularly the Fl head; determining spatial coordinates of the at least a portion of the ATP synthase complex; electronically screening stored spatial coordinates of candidate compounds against the spatial coordinates of the at least a portion of the ATP synthase complex; identifying a compound that is substantially similar to the at least a portion of the ATP synthase complex; and synthesizing the identified compound.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • the present invention provides pharmaceutical compositions comprising an effective amount of a compound having a three-dimensional structure corresponding to atomic coordinates of at least a portion of a mitochondrial ATP synthase complex.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • the present invention provides methods and systems for identifying mitochondrial ATP synthase complex modulators comprising: a processor; and a processor readable storage medium in communication with the processor readable storage medium comprising the atomic coordinates of at least a portion of a mitochondrial ATP synthase complex.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and (oligomycin- sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • the present invention provides mitochondrial ATP synthase complex binding compounds comprising a molecule having a three-dimensional structure corresponding to atomic coordinates derived from at least a portion of an atomic model of the mitochondrial ATP synthase complex.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • X- ray crystallography is an established, well-studied technique that provides what can be best described as a three-dimensional picture of what a molecule looks like in a crystal.
  • scientists have used crystallography to solve the crystal structures for many biologically important molecules.
  • Many classes of biomolecules can be studied by crystallography, including, but not limited to, proteins, DNA, RNA and viruses.
  • Advantageous therapeutic embodiments would therefore comprise therapeutic and/or diagnostic agents based on or derived from the three-dimensional crystal structure of mitochondrial ATP synthase complex including novel binding sites that have one or more than one of the functional activities of mitochondrial ATP synthase complex.
  • the ATP synthase complex may include its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein), or the ATP synthase complex may include the b subunit and the OSCP subunit, or the ATP synthase complex may include the b subunit or the ATP synthase complex may include the OSCP subunit.
  • a "binding site” refers to a region of a molecule or molecular complex that, as a result of its shape and charge potential, favorably interacts or associates with another agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, antibody or drug) via various covalent and/or non-covalent binding forces.
  • the terms "bind” and "binding" when used to describe the interaction of a ligand with a binding site or a group of amino acids means that the binding site or group of amino acids are capable of forming a covalent or non-covalent bond or bonds with the ligand.
  • the binding between the ligand and the binding site or amino acid(s) is non-covalent.
  • Such a non-covalent bond includes a hydrogen bond, an electrostatic bond, a van der Waals bond or the like.
  • the binding of the ligand to the binding site may also be characterized by the ability of the ligand to co-crystallize with mitochondrial ATP synthase complex within the binding pocket of the instant invention.
  • binding when referring to the interaction of a ligand with the binding site of the instant invention includes the covalent or non-covalent interactions of the ligand with all or some of the amino acid residues comprising the binding site.
  • an embodiment of the invention provides protein crystals of mitochondrial ATP synthase complex complexed with a ligand bound to the ligand binding site disclosed herein and methods for making mitochondrial ATP synthase complex or a mitochondrial ATP synthase complex homolog.
  • Dexpramipexole is demonstrated to bind preferentially to the b and OSCP subunits of the ATP synthase complex, defining one or more binding sites that may serve as a substrate for the elucidation of the crystal structure of the protein-ligand interaction site(s).
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit.
  • the ATP synthase complex includes the b subunit.
  • the ATP synthase complex includes the OSCP subunit.
  • the crystals provide means to obtain atomic modeling information of the specific amino acids and their atoms forming the binding site and that interact with molecules e.g., ligands or binding partners that bind to the mitochondrial ATP synthase complex, via the binding site.
  • the crystals also provide modeling information regarding the protein-ligand interaction, as well as the structure of ligands bound thereto.
  • the mitochondrial ATP synthase complex crystal or a mitochondrial ATP synthase complex homolog according to the present invention can be obtained by crystallizing it with a material or compound or molecule which binds to the herein disclosed binding site of the mitochondrial ATP synthase complex.
  • the ATP synthase complex includes its nine (9) subunits: the alpha (a), beta ( ⁇ ), b, c, delta ( ⁇ ), d, epsilon ( ⁇ ), gamma ( ⁇ ), and OSCP (oligomycin-sensitivity conferring protein).
  • the ATP synthase complex includes the b subunit and the OSCP subunit. In certain embodiments, the ATP synthase complex includes the b subunit. In certain embodiments, the ATP synthase complex includes the OSCP subunit.
  • crystalline compositions of this invention are capable of diffracting X-rays to a resolution of better than about 3.5 A, and, in some embodiments, to a resolution of about 2.6 A or better, and even more preferably to a resolution of about 2.0 A or better, and are useful for determining the three-dimensional structure of the material. (The smaller the number of angstroms, the better the resolution.)
  • the present invention provides the three-dimensional structure of human mitochondrial ATP synthase complex as well as the identification and characterization of a binding site there within.
  • the identification of this site permits design and identification of compounds that bind to the ligand binding site and modulate mitochondrial ATP synthase complex related activities.
  • the compounds include inhibitors which specifically inhibit cell proliferation.
  • the 3 -dimensional structure of the complex may be determined at high enough resolution (over 0.28 nm) using X-ray crystallographic methods. The information gained therefrom e.g., about the interaction between — and the inhibitor obtained from this can then be used to modify the inhibitor and to increase the affinity of the inhibitor for the ligand binding site of mitochondrial ATP synthase complex.
  • those atoms considered to be involved in binding to the ligand binding site of mitochondrial ATP synthase complex disclosed herein can be mutated by exchanging one or more of the amino acid residues in the ligand binding site or in the motor domain that eventually effects the function of mitochondrial ATP synthase complex on the underlying cell.
  • the coordinates from the X-ray defraction patterns can be either analyzed directly to provide the three-dimensional structure (if of sufficiently high resolution).
  • the atomic coordinates for the crystallized mitochondrial ATP synthase complex, as provided herein, can be used for structure determination.
  • the X-ray diffraction patterns obtained by methods of the present invention can be provided on computer readable media, and used to provide electron density maps.
  • the electron density maps provided by analysis of the X-ray coordinates of mitochondrial ATP synthase complex complexed with Compound X, may then be fitted using suitable computer algorithms to generate secondary, tertiary and/or quaternary structures and/or domains of mitochondrial ATP synthase complex, which structures and/or domains are then used to provide an overall three-dimensional structure, as well as binding and/or active sites of mitochondrial ATP synthase complex.
  • renin As an example, the structure of renin has been modeled using the tertiary structure of endothiapepsin as a starting point for the derivation. Model building of cercarial elastase and tophozoite cysteine protease were each built from known serine and cysteine proteases that have less than 35% sequence identity. The resultant models were used to design inhibitors in the low micromolar range. (Proc. Natl. Acad. Sci. 1993, 90, 3583).
  • tertiary structure determination that do not rely on X-ray diffraction techniques and thus do not require crystallization of the protein, such as NMR techniques, are simplified if a model of the structure is available for refinement using the additional data gathered by the alternative technique.
  • knowledge of the tertiary structure of the mitochondrial ATP synthase complex binding site provides a significant window to the structure of the other family members.
  • an embodiment of this invention envisions use of atomic coordinates of mitochondrial ATP synthase complex, or fragment, analog or variant thereof, to model a protein.
  • One skilled in the relevant art may use conventional molecular modeling methods to identify a ligand binding site of a mitochondrial ATP synthase complex of another species.
  • coordinates provided by the present invention may be used to characterize a three-dimensional structure of the target mitochondrial ATP synthase complex liganded or unliganded.
  • such a skilled artisan may, from such a structure, computationally visualize a putative binding site and identify and characterize other features based upon the coordinates provided herein.
  • Such putative ligand binding sites may be further refined using chemical shift perturbations of spectra generated from various and distinct mitochondrial ATP synthase complex complexes, e.g.
  • the NMR data from the molecular complex whose structure is unknown can then be compared to the structure data obtained from the mitochondrial ATP synthase complex of the present invention.
  • 2D, 3D and 4D isotope filtering, editing and triple resonance NMR techniques can be used to conform the 3D structure described herein for the mitochondrial ATP synthase complex complexes to the NMR data from unknown target molecular complex.
  • molecular replacement may be used to conform the 3D structure of the present invention to X-ray diffraction data from crystals of the unknown target molecular complex.
  • mitochondrial ATP synthase complex analogs in which the overall mitochondrial ATP synthase complex structure is not changed, but change does affect biological activity
  • biological activity being used here in its broadest sense to denote function
  • the three dimensional (3D) surface structure of ATP synthase can be used as a target for predicting drug design.
  • modulating compounds can be found through computer assisted searches of databases.
  • Compounds which contain the best predicted fit can then be visually inspected and tested under in vitro and in vivo conditions.
  • This method also allows for the tinkering of the compound's structure to allow for optimal binding capacity, i.e., by testing the activity of analogs of the identified compounds in in vitro assays.
  • the present invention provides a method for computational processing of a database containing three-dimensional structures of a large number of chemical compounds to identify compounds having high predicted binding affinity to a host molecule.
  • the predicted binding affinity is validated through in vitro testing.
  • One or more of the compounds having a binding affinity validated in vitro are further tested in vivo to provide a group of pharmacophores capable of having therapeutic activity involving the host molecule.
  • Computationally predicting a compound's binding affinity to a host molecule involves utilizing the three dimensional (3-D) structures of the host and the compound.
  • the 3-D structure of the compound is obtained from a database of chemical compounds.
  • the 3-D structure of the host protein can also be obtained from a protein database.
  • the invention includes a method for modeling the 3-D structure of the host protein, when such structure is not available.) Modeling the 3-D structure of the host protein includes obtaining the primary and secondary structures of the protein.
  • BLAST can be accessed at http://www.ncbi.nlm.nih.gov/BLAST- /.
  • the methodology utilized in BLAST is described in "Protein sequence similarity searches using patterns as seeds", by Zheng Zhang, Alejandro A. Schffer, Webb Miller, Thomas L. Madden, David J. Lipman, Eugene V. Koonin, and Stephen F. Altchsul (1998), Nucleic Acids Res. 26:3986-3990, the contents of which are incorporated herein by reference in their entirety.
  • a template 3-D structure of the host protein is obtained through the program MODELLER.
  • MODELLER can be obtained from Professor Andrej Sali, the Rockefeller University, 1230 York Avenue, New York, N.Y. 10021-6399.
  • the methodology utilized in MODELLER is described in "Evaluation of comparative protein modeling by MODELLER” by Sali, A., Potterton, L., Yuan, F., van Vlijmen, H., & arplus, M. (1995). Proteins, 23, 318-326, the contents of which are incorporated herein by reference in their entirety.
  • each atom in of the backbone of the protein is assigned a position corresponding to the equivalent backbone atom of the homologous protein.
  • each atom of a side chain of the host protein having an equivalent side chain in the homologous protein is assigned the position corresponding to the position of the atom in the equivalent side chain of the homologous protein.
  • the atom positions for the side chains not having an equivalent in the homologous protein are determined by constructing the side chain according to preferred internal coordinates and attaching the side chain to the backbone of the host protein.
  • the template structure thus obtained is refined by minimizing the internal energy of the template protein.
  • the positions of the atoms of the side chains having no equivalents in the homologous protein are adjusted while keeping the rest of the atoms of the template protein in a fixed position. This allows the atoms of the constructed side chains to adapt their positions to the part of the template structure determined by homology.
  • the full template structure is then minimized (relaxed) by allowing all the atoms to move. Relaxing the template 3-D structure of the protein eliminates unfavorable contacts between the atoms of the protein and reduces the strain in the template 3-D structure.
  • the minimization of the energy function associated with the template structure can be performed by any minimization technique.
  • a minimization technique involves simulated annealing. This technique is incorporated in numerous commercial and non-commercial computer programs.
  • One such computer program is included in the software package CHARMM.
  • CHARMM can be obtained either from Dr. Martin Karplus at the Harvard University for academic users or from the Molecular Simulation Inc., San Diego, Calif.
  • the simulated annealing methodology incorporated in CHARMM is described in "A program for macromolecular energy minimization, and dynamics calculations" by Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M., J. Comp. Chem. 1 (1983) 187, the contents of which are incorporated herein by reference in their entirety.
  • a host-guest complex is formed by disposing a compound from the database in a receptor site of the protein.
  • the structure of the host-guest complex is defined by the position occupied by each atom in the complex in a three dimensional referential.
  • the position of each atom is defined by a set of three coordinates in the referential.
  • the structure of the host-guest complex is incorporated in a computer program capable of determining the degree of geometrical fit between the guest and the host in the complex. Programs based on shape complementarity can effectively rank guest-host complexes based on the geometrical fit between the host and the guest.
  • a program for ranking guest-host complexes based on the geometrical fit is provided in the software package DOCK.
  • DOCK can be obtained from Dr. Irwin Kuntz at the Department of Pharmaceutical Chemistry, University of California at San Francisco, USA.
  • a group of compounds is extracted from the compound database for further processing based on their geometry fit rank.
  • the compounds in the group have a guest-host complex geometrical fit of a predetermined rank or higher.
  • the number of compounds in the geometry fit group is generally a small fraction of the total number of compounds in the database
  • a predicted binding affinity to the receptor site of the host protein is determined by minimizing an energy function describing the interactions between the atoms of the compound and those of the protein.
  • the minimization of the energy function is conducted by changing the position of the compound such that a guest- host complex structure corresponding to a minimum of the energy function is obtained.
  • the energy function includes energy terms describing non-bonded interactions between the atoms of the compound and those of the protein.
  • the non-bonded energy terms include a term for atom-atom Van der Waals interactions and a term for charge-charge electrostatic interactions.
  • the energy function does not include constraints on torsional degrees of freedom of the compound which provides greater flexibility in changing the position and conformation of the compound in the receptor site of the protein. A minimum energy value is obtained for each compound-protein complex.
  • Allowing for torsional flexibility in refining the structure of the complex greatly enhances the accuracy of the predicted binding energy of the complex.
  • a flexible compound can adopt a larger number of conformations inside the receptor site, thus allowing for probing a larger number of complex structures.
  • Increasing the number of probable complex structures increases the probability of identifying a global minimum of the energy function. That is, a minimum having an energy lower than the energy associated with one or more other identified minima of the energy function (local minima). Identifying a global minimum for a given complex is greatly advantageous in that a more accurate predicted binding affinity is obtained for the complex.
  • Increasing the accuracy of the predicted binding affinity increases the accuracy in energy based discrimination between the compounds of the geometry fit group, thus providing the best candidates for in vitro testing.
  • MCDOCK A Monte Carlo simulation approach to the molecular docking problem
  • MCDOCK provides a minimization method based on a non-conventional Monte Carlo simulation technique which allows greater probability to reach a global energy minimum.
  • the program only constrains the bonds and bond angles describing the structure of the guest host complex. Otherwise, the atoms are allowed to move freely in a force field determined by an energy function formed by Van der Waals and electrostatic terms only. This flexibility allows the guest to adopt various conformations within the receptor site of the host and thus explore a larger portion of the receptor site. This in turn allows the exploration of global minima, which improves the equality of the energy based binding affinity prediction.
  • the compounds in the geometry fit group are processed through MCDOCK such that for each compound, a compound-protein complex of minimum "MCDOCK" energy is determined.
  • the compounds are then ranked according to the minimum energy obtained.
  • a subgroup of compounds associated with complexes having a minimum energy lower than a predetermined energy value is formed.
  • the number of compounds in the subgroup is also a small fraction of the total number of compounds in the geometry fit group.
  • the binding information associated with each compound in the subgroup is further refined by displaying on a computer screen an image of the complex structure of minimum energy. Displaying the compound-protein complex is conducted through one of the conventional chemical structure graphic visualization tools.
  • a graphic visualization tool is provided in the software package QUANTA (MOLECULAR SIMULATIONS, San Diego, Calif.).
  • the displayed complexes are visually examined to form a group of candidate compounds for in vitro testing.
  • the complexes are inspected for visual determination of the quality of docking of the compound into the receptor site of the protein.
  • Visual inspection provides an effective basis for identifying compounds for in vitro testing. It should be noted that such visual inspection is impractical without the effective pruning of the compounds of the initial database provided by the pruning based on the combination of the geometry fit and complex energy minimization. Therefore, the number of compounds in the group discarded in the visual pruning step is much smaller than the number of compounds discarded in the geometry fit and energy based pruning
  • Target binding assays Methods for determining whether a compound binds to or interacts with to a particular target, i.e., target binding assays are well known in the art. In particular, this can be effected by use of competition assays. In general, this will involve providing a source of the particular receptor, a moiety known to interact with such receptor, e.g., peptide, and a compound, the receptor binding of which is to be tested. Compounds which bind the receptor will inhibit the binding of the other moiety, e.g., peptide, that is known to specifically bind said receptor.
  • compounds that modulate ATP synthase activity may be useful in treating diseases and conditions associated with or involving decreased mitochondrial function or mitochondrial dysfunction.
  • compounds that increase mitochondrial bioenergetic efficiency may be useful in treating diseases and conditions associated with or involving decreased mitochondrial function or mitochondrial dysfunction.
  • such diseases and conditions include, but are not limited to, age-related macular degeneration, type II diabetes, skin diseases and disorders, coronary and cardiovascular diseases and disorders, inflammatory disorders and neurodegenerative diseases.
  • compounds that modulate ATP synthase activity may be useful in treating a neurodegenerative disease.
  • neurodegenerative diseases include Huntington's Chorea, metabolically induced neurological damage, Alzheimer's disease, senile dementia, age associated cognitive dysfunction, vascular dementia, multi-infarct dementia, Lewy body dementia, neurodegenerative dementia, neurodegenerative movement disorder, ataxia, Friedreich's ataxia, multiple sclerosis, spinal muscular atrophy, primary lateral sclerosis, seizure disorders, motor neuron disorder or disease, inflammatory demyelinating disorder, Parkinson's disease, amyotrophic lateral sclerosis (ALS), hepatic encephalopathy, and chronic encephalitis.
  • the compositions and methods of the invention may be used to treat nearly any individual exhibiting symptoms of a neurological disease or susceptible to such diseases.
  • the amount of the compound may vary.
  • the amount of the compound may be from about 25 mg to about 1,000 mg, about 50 mg to about 1,000 mg, from about 100 mg to about 1 ,000 mg, from about 300 mg to about 1,000 mg, from about 500 mg to about 1 ,000 mg, and in certain embodiments, the amount may be from about 60 mg to about 300 mg.
  • the amount of the compound administered per day, the therapeutically effective amount per day per kg may be from about 2 mg/kg/day to about 1000 mg/kg/day, from about 4 mg/kg/day to about 1000 mg/kg/day, from about 2 mg/kg/day to about 500 mg/kg/day, from about 4 mg/kg/day to about 500 mg/kg/day, from about 2 mg/kg/day to about 200 mg/kg/day, or from about 4 mg/kg/day to about 200 mg/kg/day, and in other embodiments, the amount may be from about from about 1 mg/kg/day to about 100 mg/kg/day, from about 2 mg/kg/day to about 50 mg/kg/day, or from about 2 mg/kg/day to about 10 mg/kg/day.
  • the daily dose administered to a patient in various embodiments, may from about 100 mg/day to about 1000 mg/day, about 150 mg/day to 500 mg/day, and in particular embodiments, from about 150 mg/day to about 300 mg/
  • the invention is directed to a pharmaceutical composition comprising a compound, as defined above, and a pharmaceutically acceptable carrier or diluent, or an effective amount of a pharmaceutical composition comprising a compound as defined above.
  • the compounds of the present invention can be administered in the conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants.
  • modes of administration for the compounds of the present invention can be, but are not limited to, sublingual, injectable (including short- acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.
  • Specific modes of administration will depend on the indication.
  • the selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response.
  • the amount of compound to be administered is that amount which is therapeutically effective.
  • the dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular animal treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).
  • compositions containing the compounds of the present invention and a suitable carrier can be solid dosage forms which include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limited to, solutions, powders, fluid emulsions, fluid suspensions, semisolids, ointments, pastes, creams, gels and jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the present invention.
  • the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like.
  • pharmaceutically acceptable diluents fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like.
  • the means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) can be consulted
  • the compounds of the present invention can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • the compounds can be administered by continuous infusion subcutaneously over a period of about 15 minutes to about 24 hours.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the compounds can be formulated readily by combining these compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP).
  • disintegrating agents can be added, such as, but not limited to, the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores can be provided with suitable coatings.
  • suitable coatings can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • the compounds of the present invention can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds of the present invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compounds of the present invention for example, can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.
  • compositions of the compounds also can comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.
  • the compounds of the present invention can also be administered in combination with other active ingredients, such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.
  • active ingredients such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.
  • the disintegrant component comprises one or more of croscarmellose sodium, carmellose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, an ion exchange resin, an effervescent system based on food acids and an alkaline carbonate component, clay, talc, starch, pregelatinized starch, sodium starch glycolate, cellulose floe, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, a metal carbonate, sodium bicarbonate, calcium citrate, or calcium phosphate.
  • the diluent component comprises one or more of mannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodium starch glycolate, pregelatinized starch, a calcium phosphate, a metal carbonate, a metal oxide, or a metal aluminosilicate.
  • the optional lubricant component when present, comprises one or more of stearic acid, metallic stearate, sodium stearyl fumarate, fatty acid, fatty alcohol, fatty acid ester, glyceryl behenate, mineral oil, vegetable oil, paraffin, leucine, silica, silicic acid, talc, propylene glycol fatty acid ester, polyethoxylated castor oil, polyethylene glycol, polypropylene glycol, polyalkylene glycol, polyoxyethylene-glycerol fatty ester, polyoxyethylene fatty alcohol ether, polyethoxylated sterol, polyethoxylated castor oil, polyethoxylated vegetable oil, or sodium chloride.
  • alginic acid refers to a naturally occurring hydrophilic colloidal polysaccharide obtained from the various species of seaweed, or synthetically modified polysaccharides thereof.
  • sodium alginate refers to a sodium salt of alginic acid and can be formed by reaction of alginic acid with a sodium containing base such as sodium hydroxide or sodium carbonate.
  • potassium alginate refers to a potassium salt of alginic acid and can be formed by reaction of alginic acid with a potassium containing base such as potassium hydroxide or potassium carbonate.
  • calcium alginate refers to a calcium salt of alginic acid and can be formed by reaction of alginic acid with a calcium containing base such as calcium hydroxide or calcium carbonate.
  • Suitable sodium alginates, calcium alginates, and potassium alginates include, but are not limited to, those described in R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
  • Suitable sodium alginates include, but are not limited to, Kelcosol (available from ISP), Kelfone LVCR and HVCR (available from ISP), Manucol (available from ISP), and Protanol (available from FMC Biopolymer).
  • calcium silicate refers to a silicate salt of calcium.
  • calcium phosphate refers to monobasic calcium phosophate, dibasic calcium phosphate or tribasic calcium phosphate.
  • Cellulose, cellulose floe, powdered cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, carboxyethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, ethylcellulose, methylcellulose, carboxymethylcellulose sodium, and carboxymethyl cellulose calcium include, but are not limited to, those described in R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
  • cellulose refers to natural cellulose.
  • cellulose also refers to celluloses that have been modified with regard to molecular weight and/or branching, particularly to lower molecular weight.
  • cellulose further refers to celluloses that have been chemically modified to attach chemical functionality such as carboxy, hydroxyl, hydroxyalkylene, or carboxyalkylene groups.
  • carboxyalkylene refers to a group of formula -alkylene-C(0)OH, or salt thereof.
  • hydroxyalkylene refers to a group of formula -alkylene-OH.
  • Suitable powdered celluloses for use in the invention include, but are not limited to Arbocel (available from JRS Pharma), Sanacel (available from CFF GmbH), and Solka-Floc (available from International Fiber Corp.).
  • Suitable microcrystalline celluloses include, but are not limited to, the Avicel pH series (available from FMC Biopolymer), Celex (available from ISP), Celphere (available from Asahi Kasei), Ceolus KG (available from Asahi Kasei), and Vivapur (available from JRS Pharma).
  • silicified microcrystalline cellulose refers to a synergistic intimate physical mixture of silicon dioxide and microcrystalline cellulose. Suitable silicified microcrystalline celluloses include, but are not limited to, ProSolv (available from JRS Pharma).
  • carboxymethylcellulose sodium refers to a cellulose ether with pendant groups of formula Na + -C(0)-CH 2 -, attached to the cellulose via an ether linkage.
  • Suitable carboxymethylcellulose sodium polymers include, but are not limited to, Akucell (available from Akzo Nobel), Aquasorb (available from Hercules), Blanose (available from Hercules), Finnfix (available from Noviant), Nymel (available from Noviant), and Tylose CB (available from Clariant).
  • carboxymethylcellulose calcium refers to a cellulose ether with a pendant groups of formula -CH 2 -0-C(0)-0 " 1 ⁇ 2 Ca 2+ , attached to the cellulose via an ether linkage.
  • carboxymethylcellulose refers to a cellulose ether with pendant carboxymethyl groups of formula HO-C(0)-CH 2 -, attached to the cellulose via an ether linkage.
  • Suitable carboxymethylcellulose calcium polymers include, but are not limited to, Nymel ZSC (available from Noviant).
  • carboxyethylcellulose refers to a cellulose ether with pendant carboxymethyl groups of formula HO-C(0)-CH 2 -CH 2 -, attached to the cellulose via an ether linkage.
  • hydroxyethylcellulose refers to a cellulose ether with pendant hydroxyethyl groups of formula HO-CH 2 -CH 2 -, attached to the cellulose via an ether linkage.
  • Suitable hydroxyethylcelluloses include, but are not limited to, Cellosize HEC (available from DOW), Natrosol (available from Hercules), and Tylose PHA (available from Clariant).
  • methylhydroxyethylcellulose refers to a cellulose ether with pendant methyloxyethyl groups of formula CH -0-CH 2 -CH 2 -, attached to the cellulose via an ether linkage.
  • Suitable methylhydroxyethylcelluloses include, but are not limited to, the Culminal MHEC series (available from Hercules), and the Tylose series (available from Shin Etsu).
  • hydroxypropylcellulose refers a cellulose that has pendant hydroxypropoxy groups, and includes both high- and low-substituted hydroxypropylcellulose. In some embodiments, the hydroxypropylcellulose has about 5% to about 25% hydroxypropyl groups.
  • Suitable hydroxypropylcelluloses include, but are not limited to, the Klucel series (available from Hercules), the Methocel series (available from Dow), the Nisso HPC series (available from Nisso), the Metolose series (available from Shin Etsu), and the LH series, including LHR-11, LH-21, LH-31, LH-20, LH-30, LH-22, and LH-32 (available from Shin Etsu).
  • methyl cellulose refers to a cellulose that has pendant methoxy groups. Suitable methyl celluloses include, but are not limited to Culminal MC (available from Hercules).
  • ethyl cellulose refers to a cellulose that has pendant ethoxy groups. Suitable ethyl celluloses include, but are not limited to Aqualon (available from Hercules).
  • carboxymethylcellulose calcium refers to a crosslinked polymer of carboxymethylcellulose calcium.
  • croscarmellose sodium refers to a crosslinked polymer of carboxymethylcellulose sodium.
  • crospovidone refers to a crosslinked polymer of polyvinylpyrrolidone. Suitable crospovidone polymers include, but are not limited to Polyplasdone XL- 10 (available from ISP) and Kollidon CL and CL-M (available from BASF).
  • crosslinked poly(acrylic acid) refers to a polymer of acrylic acid which has been crosslinked.
  • the crosslinked polymer may contain other monomers in addition to acrylic acid. Additionally, the pendant carboxy groups on the crosslinked polymer may be partially or completely neutralized to form a pharmaceutically acceptable salt of the polymer.
  • the crosslinked poly(acrylic acid) is neutralized by ammonia or sodium hydroxide.
  • Suitable crosslinked poly(acrylic acid) polymers include, but are not limited to, the Carbopol series (available from Noveon).
  • an effervescent system based on food acids and an alkaline carbonate component refers to a excipient combination of food acids and alkaline carbonates that releases carbon dioxide gas when administered.
  • Suitable effervescent systems are those that those utilizing food acids (such as citric acid, tartaric acid, malic acid, fumaric acid, lactic acid, adipic acid, ascorbic acid, aspartic acid, erythorbic acid, glutamic acid, and succinic acid) and an alkaline carbonate component (such as sodium bicarbonate, calcium carbonate, magnesium carbonate, potassium carbonate, ammonium carbonate, etc.).
  • the term "fatty acid”, employed alone or in combination with other terms, refers to an aliphatic acid that is saturated or unsaturated. In some embodiments, the fatty acid in a mixture of different fatty acids. In some embodiments, the fatty acid has between about eight to about thirty carbons on average. In some embodiments, the fatty acid has about eight to about twenty-four carbons on average. In some embodiments, the fatty acid has about twelve to about eighteen carbons on average.
  • Suitable fatty acids include, but are not limited to, stearic acid, lauric acid, myristic acid, erucic acid, palmitic acid, palmitoleic acid, capric acid, caprylic acid, oleic acid, linoleic acid, linolenic acid, hydroxystearic acid, 12- hydroxystearic acid, cetostearic acid, isostearic acid, sesquioleic acid, sesqui-9-octadecanoic acid, sesquiisooctadecanoic acid, benhenic acid, isobehenic acid, and arachidonic acid, or mixtures thereof.
  • fatty acid ester refers to a compound formed between a fatty acid and a hydroxyl containing compound.
  • the fatty acid ester is a sugar ester of fatty acid.
  • the fatty acid ester is a glyceride of fatty acid.
  • the fatty acid ester is an ethoxylated fatty acid ester.
  • the term "fatty alcohol”, employed alone or in combination with other terms, refers to an aliphatic alcohol that is saturated or unsaturated. In some embodiments, the fatty alcohol in a mixture of different fatty alcohols. In some embodiments, the fatty alcohol has between about eight to about thirty carbons on average. In some embodiments, the fatty alcohol has about eight to about twenty-four carbons on average. In some embodiments, the fatty alcohol has about twelve to about eighteen carbons on average.
  • Suitable fatty alcohols include, but are not limited to, stearyl alcohol, lauryl alcohol, palmityl alcohol, palmitolyl acid, cetyl alcohol, capryl alcohol, caprylyl alcohol, oleyl alcohol, linolenyl alcohol, arachidonic alcohol, behenyl alcohol, isobehenyl alcohol, selachyl alcohol, chimyl alcohol, and linoleyl alcohol, or mixtures thereof.
  • the term "ion-exchange resin” refers to an ion-exchange resin that is pharmaceutically acceptable and that can be weakly acidic, weakly basic, strongly acidic or strongly basic.
  • Suitable ion-exchange resins include, but are not limited to AmberliteTM IRP64, IRP88 and IRP69 (available from Rohm and Haas) and DuoliteTM API 43 (available from Rohm and Haas).
  • the ion-exchange resin is a crosslinked polymer resin comprising acrylic acid, methacrylic acid, or polystyrene sulfonate, or salts thereof.
  • the ion-exchange resin is polacrilex resin, polacrilin potassium resin, or cholestyramine resin.
  • Suitable mannitols include, but are not limited to, PharmMannidex (available from Cargill), Pearlitol (available from Roquette), and Mannogem (available from SPI Polyols).
  • metal aluminosilicate refers to any metal salt of an aluminosilicate, including, but not limited to, magnesium aluminometasilicate.
  • Suitable magnesium aluminosilicates include, but are not limited to Neusilin (available from Fuji Chemical), Pharmsorb (available from Engelhard), and Veegum (available from R.T. Vanderbilt Co., Inc.).
  • the metal aluminosilicate is bentonite.
  • metal carbonate refers to any metallic carbonate, including, but not limited to sodium carbonate, calcium carbonate, and magnesium carbonate, and zinc carbonate.
  • metal oxide refers to any metallic oxide, including, but not limited to, calcium oxide or magnesium oxide.
  • the term "metallic stearate” refers to a metal salt of stearic acid.
  • the metallic stearate is calcium stearate, zinc stearate, or magnesium stearate. In some embodiments, the metallic stearate is magnesium stearate.
  • mineral oil refers to both unrefined and refined (light) mineral oil. Suitable mineral oils include, but are not limited to, the AvatechTM grades (available from Avatar Corp.), DrakeolTM grades (available from Penreco), SiriusTM grades (available from Shell), and the CitationTM grades (available from Avater Corp.).
  • polyethoxylated castor oil refers to a compound formed from the ethoxylation of castor oil, wherein at least one chain of polyethylene glycol is covalently bound to the castor oil.
  • the castor oil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylated castor oil include, but are not limited to polyoxyl castor oil, hydrogenated polyoxyl castor oil, mcrogolglyceroli ricinoleas, macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl 40 hydrogenated castor oil.
  • Suitable polyethoxylated castor oils include, but are not limited to, the NikkolTM HCO series (available from Nikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, and HC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenated castor oil, and polyethylene glycol-60 hydrogenated castor oil, EmulphorTM EL-719 (castor oil 40 mole-ethoxylate, available from Stepan Products), the CremophoreTM series (available from BASF), which includes Cremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-60 hydrogenated castor oil, and polyethylene glycol-35 hydrogenated castor oil, respectively), and the Emulgin® RO and HRE series (available from Cognis PharmaLine).
  • Other suitable polyoxyethylene castor oil derivatives include those listed in R. C. Rowe and P. J
  • polyethoxylated sterol refers to a compound, or mixture of compounds, derived from the ethoxylation of sterol molecule.
  • Suitable polyethoyxlated sterols include, but are not limited to, PEG-24 cholesterol ether, SolulanTM C- 24 (available from Amerchol); PEG-30 cholestanol, NikkolTM DHC (available from Nikko); Phytosterol, GENEROLTM series (available from Henkel); PEG-25 phyto sterol, NikkolTM BPSH-25 (available from Nikko); PEG-5 soya sterol, NikkolTM BPS-5 (available from Nikko); PEG- 10 soya sterol, NikkolTM BPS- 10 (available from Nikko); PEG-20 soya sterol, NikkolTM BPS-20 (available from Nikko); and PEG-30 soya sterol, NikkolTM BPS-30 (available from NikkolTM
  • polyethoxylated vegetable oil refers to a compound, or mixture of compounds, formed from ethoxylation of vegetable oil, wherein at least one chain of polyethylene glycol is covalently bound to the the vegetable oil.
  • the fatty acids has between about twelve carbons to about eighteen carbons.
  • the amount of ethoxylation can vary from about 2 to about 200, about 5 to 100, about 10 to about 80, about 20 to about 60, or about 12 to about 18 of ethylene glycol repeat units.
  • the vegetable oil may be hydrogenated or unhydrogenated.
  • Suitable polyethoxylated vegetable oils include but are not limited to, CremaphorTM EL or RH series (available from BASF), EmulphorTM EL-719 (available from Stepan products), and EmulphorTM EL-620P (available from GAF).
  • polyethylene glycol refers to a polymer containing ethylene glycol monomer units of formula -0-CH 2 -CH 2 -. Suitable polyethylene glycols may have a free hydroxyl group at each end of the polymer molecule, or may have one or more hydroxyl groups etherified with a lower alkyl, e.g., a methyl group. Also suitable are derivatives of polyethylene glycols having esterifiable carboxy groups. Polyethylene glycols useful in the present invention can be polymers of any chain length or molecular weight, and can include branching. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 9000.
  • the average molecular weight of the polyethylene glycol is from about 200 to about 5000. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 900. In some embodiments, the average molecular weight of the polyethylene glycol is about 400.
  • Suitable polyethylene glycols include, but are not limited to polyethylene glycol-200, polyethylene glycol-300, polyethylene glycol-400, polyethylene glycol-600, and polyethylene glycol-900. The number following the dash in the name refers to the average molecular weight of the polymer. In some embodiments, the polyethylene glycol is polyethylene glycol-400.
  • Suitable polyethylene glycols include, but are not limited to the CarbowaxTM and CarbowaxTM Sentry series (available from Dow), the LipoxolTM series (available from Brenntag), the LutrolTM series (available from BASF), and the PluriolTM series (available from BASF).
  • polyoxyethylene-alkyl ether refers to a monoalkyl or dialkylether of polyoxyethylene, or mixtures thereof.
  • the polyoxyethylene-alkyl ether is a polyoxyethylene fatty alcohol ether.
  • polyoxyethylene fatty alcohol ether refers to an monoether or diether, or mixtures thereof, formed between polyethylene glycol and a fatty alcohol.
  • Fatty alcohols that are useful for deriving polyoxyethylene fatty alcohol ethers include, but are not limited to, those defined herein.
  • the polyoxyethylene portion of the molecule has about 2 to about 200 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 2 to about 100 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 4 to about 50 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 4 to about 30 oxyethylene units.
  • the polyoxyethylene fatty alcohol ether comprises ethoxylated stearyl alcohols, cetyl alcohols, and cetylstearyl alcohols (cetearyl alcohols).
  • Suitable polyoxyethylene fatty alcohol ethers include, but are not limited to, the BrijTM series of surfactants (available from Uniqema), which includes Brij 30, 35, 52, 56, 58, 72, 76, 78, 93Veg, 97, 98, and 721, the CremophorTM A series (available from BASF), which includes Cremophor A6, A20, and A25, the EmulgenTM series (available from Kao Corp.), which includes Emulgen 104P, 123P, 210P, 220, 320P, and 409P, the EthosperseTM (available from Lonza), which includes Ethosperse 1A4, 1A12, TDAa6, SI 20, and G26, the EthylanTM series (available from Brenntag), which includes Ethylan D252, 253,
  • polyoxyethylene fatty alcohol ethers include, but are not limited to, polyethylene glycol (13)stearyl ether (steareth-13), polyethylene glycol (14)stearyl ether (steareth-14), polyethylene glycol (15)stearyl ether (steareth-15), polyethylene glycol (16)stearyl ether (steareth-16), polyethylene glycol (17)stearyl ether (steareth-17), polyethylene glycol (18)stearyl ether (steareth-18), polyethylene glycol (19)stearyl ether (steareth-19), polyethylene glycol (20)stearyl ether (steareth-20), polyethylene glycol (12)isostearyl ether (isosteareth-12), polyethylene glycol (13)isostearyl ether (isosteareth-13), polyethylene glycol (14)isostearyl ether (isosteareth-14), polyethylene glycol (15)isostearyl ether (isosteareth-15), polyethylene glycol (16)isostearyl ether (isoste
  • polyethylene glycol refers to the number of oxyethylene repeat units in the compound.
  • Blends of polyoxyethylene fatty alcohol ethers with other materials are also useful in the invention.
  • a non-limiting example of a suitable blend is ArlacelTM 165 or 165 VEG (available from Uniqema), a blend of glycerol monostearate with polyethylene glycol- 100 stearate.
  • Other suitable polyoxyethylene fatty alcohol ethers include those listed in R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
  • polyoxyethylene-glycerol fatty ester refers to ethoxylated fatty acid ester of glycerine, or mixture thereof.
  • the polyoxyethylene portion of the molecule has about 2 to about 200 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 2 to about 100 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 4 to about 50 oxyethylene units. In some embodiments, the polyoxyethylene portion of the molecule has about 4 to about 30 oxyethylene units.
  • Suitable polyoxyethylene-glycerol fatty esters include, but are not limited to, PEG-20 glyceryl laurate, TagatTM L (Goldschmidt); PEG-30 glyceryl laurate, TagatTM L2 (Goldschmidt); PEG- 15 glyceryl laurate, GlyceroxTM L series (Croda); PEG-40 glyceryl laurate, GlyceroxTM L series (Croda); PEG-20 glyceryl stearate, CapmulTM EMG (ABITEC), Aldo MS-20 KFG (Lonza); PEG-20 glyceryl oleate, TagatTM 0 (Goldschmidt); PEG-30 glyceryl oleate, TagatTM 02 (Goldschmidt).
  • propylene glycol fatty acid ester refers to an monoether or diester, or mixtures thereof, formed between propylene glycol or polypropylene glycol and a fatty acid.
  • Fatty acids that are useful for deriving propylene glycol fatty alcohol ethers include, but are not limited to, those defined herein.
  • the monoester or diester is derived from propylene glycol.
  • the monoester or diester has about 1 to about 200 oxypropylene units.
  • the polypropylene glycol portion of the molecule has about 2 to about 100 oxypropylene units.
  • the monoester or diester has about 4 to about 50 oxypropylene units. In some embodiments, the monoester or diester has about 4 to about 30 oxypropylene units.
  • Suitable propylene glycol fatty acid esters include, but are not limited to, propylene glycol laurates: LauroglycolTM FCC and 90 (available from Gattefosse); propylene glycol caprylates: CapryolTM PGMC and 90 (available from Gatefosse); and propylene glycol dicaprylocaprates: LabrafacTM PG (available from Gatefosse).
  • Suitable sorbitols include, but are not limited to, PharmSorbidex E420 (available from Cargill), Liponic 70-NC and 76-NC (available from Lipo Chemical), Neosorb (available from Roquette), Partech SI (available from Merck), and Sorbogem (available from SPI Polyols).
  • PharmSorbidex E420 available from Cargill
  • Liponic 70-NC and 76-NC available from Lipo Chemical
  • Neosorb available from Roquette
  • Partech SI available from Merck
  • Sorbogem available from SPI Polyols.
  • Starch, sodium starch glycolate, and pregelatinized starch include, but are not limited to, those described in R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
  • starch refers to any type of natural or modified starch including, but not limited to, maize starch (also known as corn starch or maydis amylum), potato starch (also known as solani amylum), rice starch (also known as oryzae amylum), wheat starch (also known as tritici amylum), and tapioca starch.
  • maize starch also known as corn starch or maydis amylum
  • potato starch also known as solani amylum
  • rice starch also known as oryzae amylum
  • wheat starch also known as tritici amylum
  • tapioca starch tapioca starch.
  • starch also refers to starches that have been modified with regard to molecular weight and branching.
  • starch further refers to starches that have been chemically modified to attach chemical functionality such as carboxy, hydroxyl, hydroxyalkylene, or carboxyalkylene groups.
  • carboxyalkylene refers to a group of formula -alkylene-C(0)OH, or salt thereof.
  • hydroxyalkylene refers to a group of formula -alkylene- OH.
  • Suitable sodium starch glycolates include, but are not limited to, Explotab
  • Suitable pregelatinized starches include, but are not limited to, Lycatab C and PGS (available from Roquette), Merigel (available from Brenntag), National 78-1551 (available from National Starch), Spress B820 (available from GPC), and Starch 1500 (available from Colorcon).
  • stearoyl macrogol glyceride refers to a polyglycolized glyceride synthesized predominately from stearic acid or from compounds derived predominately from stearic acid, although other fatty acids or compounds derived from other fatty acids may used in the synthesis as well.
  • Suitable stearoyl macrogol glycerides include, but are not limited to, Gelucire® 50/13 (available from Gattefosse).
  • vegetable oil refers to naturally occurring or synthetic oils, which may be refined, fractionated or hydrogenated, including triglycerides. Suitable vegetable oils include, but are not limited to castor oil, hydrogenated castor oil, sesame oil, corn oil, peanut oil, olive oil, sunflower oil, safflower oil, soybean oil, benzyl benzoate, sesame oil, cottonseed oil, and palm oil.
  • Suitable vegetable oils include commercially available synthetic oils such as, but not limited to, MiglyolTM 810 and 812 (available from Dynamit Nobel Chicals, Sweden) NeobeeTM M5 (available from Drew Chemical Corp.), AlofineTM (available from Jarchem Industries), the LubritabTM series (available from JRS Pharma), the SterotexTM (available from Abitec Corp.), SoftisanTM 154 (available from Sasol), CroduretTM (available from Croda), FancolTM (available from the Fanning Corp.), CutinaTM HR (available from Cognis), SimulsolTM (available from CJ Petrow), EmConTM CO (available from Amisol Co.), LipvolTM CO, SES, and HS-K (available from Lipo), and SterotexTM HM (available from Abitec Corp.).
  • synthetic oils such as, but not limited to, MiglyolTM 810 and 812 (available from Dynamit Nobel Chicals, Sweden) NeobeeTM M5 (available from Drew Chemical Corp.), AlofineTM (available
  • Suitable vegetable oils including sesame, castor, corn, and cottonseed oils, include those listed in R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
  • Example 1 Dexpramipexole binds to a site in Complex V, inhibiting mitochondrial leak conductance and improving cellular bioenergetic efficiency
  • Dexpramipexole (KNS-760704; (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6- benzothiazole-diamine) is a neuroprotective drug showing promising early clinical results in amyotrophic lateral sclerosis (ALS).
  • Dexpramipexole inhibited proteasome inhibitor- or calcium-induced ion conductance in rat brain-derived mitochondria, as did the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (CSA).
  • mPTP mitochondrial permeability transition pore
  • Dexpramipexole and CSA also inhibited a novel ATP-sensitive conductance recorded from sub-mitochondrial vesicles (SMVs) enriched in F1F 0 ATP synthase.
  • SMVs sub-mitochondrial vesicles
  • Dexpramipexole failed to elicit ion conductance changes in SMVs lacking functional Fl, and 14 C-dexpramipexole bound specifically to purified recombinant b and OSCP subunits of the FIFo ATP synthase.
  • Dexpramipexole maintained or increased ATP levels in neurons while oxygen consumption was decreased, indicating an increase in bioenergetic efficiency, and dexpramipexole normalized the metabolic profile of proteasome-inhibitor treated cells.
  • Dexpramipexole may act to increase the efficiency of oxidative phosphorylation in neurons at risk by inhibiting a metabolic 'leak' conductance
  • ALS is a rapidly progressive, fatal neuromuscular disease characterized by the loss of upper and lower motor neurons.
  • Mitochondrial dysfunction has been implicated in ALS, including changes in the structural integrity of mitochondria, disruption of energy metabolism and abnormal calcium (Ca 2+ ) buffering. Mitochondrial dysfunction may be common to other chronic neurodegenerative disorders (NDDs), suggesting that when critical proportions of mitochondria in specific neuronal populations become dysfunctional, neurons are put at risk. Critical functions are compromised and cell death may result if ATP synthesis cannot match cellular energy requirements.
  • NDDs chronic neurodegenerative disorders
  • the present study examined the mitochondrial actions of the candidate ALS drug dexpramipexole (KNS-760704; (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6- benzothiazole-diamine).
  • KNS-760704 (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6- benzothiazole-diamine.
  • Evidence is presented that shows a novel enhancement of the efficiency of oxidative phosphorylation by dexpramipexole resulting from the inhibition of a 'leak' conductance associated with the FIFo ATP synthase.
  • Dexpramipexole is a neuroprotective drug previously suggested to slow ALS disease progression in an open label clinical trial.
  • Dexpramipexole is the non-dopaminergic R(+) enantiomer of the dopamine agonist and Parkinson's disease therapeutic pramipexole (Mirapex ® ; (6S)-4,5,6,7-tetrahydro- N6-propyl-2,6-benzothiazole-diamine). Pramipexole is protective by a non-dopaminergic mechanism in in vitro and in vivo models of cell death and NDDs, but only at higher concentrations (>10 ⁇ ) than would be tolerated in humans. Dexpramipexole is equally protective and is tolerated at clinical doses which allow its use as a neuroprotective drug. Both enantiomers were believed to act at the level of the mitochondrion, but their specific mechanism of action remained elusive.
  • Mitochondria produce adenosine triphosphate (ATP) by oxidative phosphorylation, and the efficiency of this process can be affected by membrane currents that uncouple the electron transport system and oxidative phosphorylation.
  • Mitochondrial membrane conductances participate in the initiation of cell death but also play a major role in controlling the daily metabolic health of cells. Ion channels in dysfunctional neuronal mitochondria play a direct role in the onset of cell death in conditions such as neuronal trauma and hypoxia/ischemia, but may function differently in chronic NDDs such as ALS. Recently, pramipexole was shown to inhibit calcium (Ca2+)-induced membrane currents in rat liver mitoplasts.
  • dexpramipexole inhibited stress-induced membrane currents in brain-derived mitochondria, also inhibited similar currents in submitochondrial vesicles (SMVs), interacted with purified recombinant subunits of the FIFO ATPsynthase (complex V) while maintaining or enhancing complex V enzymatic activity.
  • Dexpramipexole did not inhibit optically-recorded calcium-induced permeability transition in liver mitochondria, but enhanced bioenergetic efficiency in neurons and other cells. These mitochondrial effects are novel, and may underlie the cellular protective effects of dexpramipexole.
  • PSI pre-treatment in rats produced currents in isolated brain mitochondria that were inhibited by dexpramipexole and CSA
  • Rats were exposed in vivo to a proteasome inhibitor (carbobenzoxyl-Ile-Glu(0- t-butyl)-Ala-leucinal; PSI) to model changes in aberrant protein accumulation that may lead to chronic mitochondrial dysfunction.
  • Mitochondria were isolated from a subcortical brain fraction after repeated dosing of rats with PSI (PSI-mitochondria, see Methods).
  • PSI-mitochondria incubated in the absence of added Ca 2+ , manifested intermediate- and large-conductance activity at a significantly higher frequency than mitochondria from control rat brains (Fig. la), and similar to the activity observed in mitochondria exposed to Ca 2+ (as described below) or previously observed in post-ischemic brain-derived mitochondria
  • dexpramipexole may inhibit the pore-forming protein complex known as the mitochondrial permeability transition pore (mPTP).
  • mPTP mitochondrial permeability transition pore
  • the mPTP participates in the initiation of some forms of cell death, although its roles in long term changes in mitochondrial function are likely to be different from its roles in more acute phenomena.
  • Addition of Ca 2+ to isolated mitochondria activates the mPTP and cyclosporine A (CSA) specifically inhibits it.
  • CSA effectively blocked channel activity in PSI-mitochondria and significantly decreased the peak membrane conductance (Fig. 2a).
  • Current that was sensitive to dexpramipexole was equally sensitive to CSA after dexpramipexole washout. Dexpramipexole- sensitive currents in PSI-mitochondria therefore share features with the mPTP, such as inhibition by CSA.
  • Dexpramipexole enhanced metabolic efficiency of cultured cells [00207] To determine if the effects of dexpramipexole measured in isolated mitochondria and SMVs result in altered mitochondrial function, including changes in ATP levels and oxygen uptake in neurons, groups of cultured hippocampal neurons (DIV 14) were treated with dexpramipexole.
  • ATP levels in neurons treated with dexpramipexole (10 ⁇ ) were increased by 11% compared to vehicle-treated cultures (Fig. 4a).
  • mitochondrial oxygen uptake contributes both to the production of ATP by oxidative phosphorylation and to counteracting 'leak' of protons which can decrease the coupling between the electron transport system and ATP production by reducing proton-motive force.
  • oxygen flux was measured in single cultured hippocampal neurons using a self-referencing oxygen-sensitive electrode (Fig. 4b,4c,4d).
  • SH-SY5Y neuroblastoma cells incubation of SH-SY5Y neuroblastoma cells in dexpramipexole (1-100 ⁇ ; 24 hr), significantly increased ATP levels, relative to control values, with maximal group increases -18%; visualized using a Seahorse ® respirometry system.
  • Dexpramipexole (30 ⁇ ) also modestly decreased basal oxygen consumption rate (OCR) in these cells.
  • SH-SY5Y cells were also incubated in galactose, a sugar that exclusively requires mitochondrial (rather than glycolytic) metabolism to produce ATP.
  • the Seahorse ® respirometry system was also used with cultured C2C12 myoblasts to determine the effects of PSI (18 hr incubation) on bioenergetic profiles.
  • C2C12 cells a CRC for cell death by PSI was established (Fig. 5a), and a PSI concentration that was near the inflection point of the killing curve (30 nM) was used to stress but not kill C2C12 cells in subsequent experiments.
  • PSI-treated or control C2C12 cells were incubated in dexpramipexole (30 ⁇ ) or control medium, and relative OCR and the extracellular acidification rate (ECAR) were measured. Under these conditions, neither ATP levels nor cell viability (Fig.
  • SMVs can hydrolyze ATP, and it was found that dexpramipexole significantly enhanced ATP hydrolysis in SMVs in a concentration-dependent manner in an assay where complex V enzymatic activity was estimated by the change in NADH signal (Fig. 6a); CSA also significantly enhanced ATP hydrolysis (Fig. 6a).
  • the ATP synthase inhibitor oligomycin effectively inhibited ATP hydrolysis in these assays.
  • Dexpramipexole inhibited PSI-induced cell death in a concentration-dependent manner when tested in SH-SY5Y neuroblastoma cells.
  • effective cytoprotective concentrations of dexpramipexole (or pramipexole) were significantly higher than the EC50S for dexpramipexole-induced mitochondrial current inhibition or enhancement of ATP synthesis.
  • concentrations producing complete elimination of these aberrant currents an EC 100
  • concentrations producing complete elimination of these aberrant currents (an EC 100), and corresponding to a 10-30 ⁇ level may be required for enhanced bioenergetic efficiency and significant cytoprotection.
  • Mitochondrial dysfunction has long been implicated in the pathogenesis of neurodegenerative disease. Proteasomal dysfunction is a potential mechanism for accumulation of undegraded proteins in mitochondria, and this has been suggested as one of many potentially stressful events coupled to the onset of neurodegeneration in Alzheimer's disease, ALS and PD. It has been demonstrated that mitochondrial stress induced by PSI (or Ca 2+ ) results in increased membrane current in mitochondrial membranes, conductance that is expressed at high levels in otherwise untreated SMVs. This membrane current constitutes a leak conductance, probably closely associated with complex V, that results in a reduction in bioenergetic efficiency.
  • PSI or Ca 2+
  • Figure 1 shows that dexpramipexole inhibited PSI-induced currents in brain- derived mitochondria.
  • the panels of Figure 1 are described as the following: Fig. la. Repeated pre-treatment of rats with PSI produced a significant increase in intermediate- and large- conductance mitochondrial ion channel activity of mitochondria isolated from subcortex, relative to mitochondria obtained from control animals.
  • PSI in this case was dosed every other day for 1 week; animals were sacrificed and mitochondria were isolated at the end of PSI dosing.
  • Fig. lb Example of a continuous patch clamp recording from a PSI- mitochondrion (see Methods) in normal recording medium, and the reversible reduction in leak conductance during the bath application of dexpramipexole (DEX).
  • Fig. Id Example of a continuous patch clamp recording from a PSI- mitochondrion (see Methods) in normal recording medium, and the reversible reduction in leak conductance during the bath application of dexpramipexole (DEX).
  • Fig. lc Group data showing that dexpramipexole (2 ⁇ ) significantly and reversibly decreased peak conduct
  • Dexpramipexole produced a concentration-dependent decrease in the mean open probability of intermediate-conductance (-500 pS) channels recorded from PSI mitochondria.
  • This example shows recordings from the same organelle-attached patch made in control medium, 2, 20 and 200 nM dexpramipexole, and during a wash to control medium; holding potential of + 80 mV. Note that in this example many closures reveal sub-conductance states. Sample recordings were obtained at steady-state for each condition.
  • Figure 2 shows that cyclosporine A (CSA) reduced peak conductance in dexpramipexole- sensitive PSI-mitochondria, and high calcium (Ca ) induced dexpramipexole- sensitive currents in normal brain mitochondria.
  • Figure 2 also shows that dexpramipexole did not inhibit mitochondrial permeability transition recorded in rat liver mitochondria.
  • Baseline optical absorbance of fresh, respiring rat liver mitochondria (measured respiratory control ratio>5) in response to 100 ⁇ Ca 2+ or alamethicin (AM) exemplifying the phenomenon of mitochondrial permeability transition in the presence of the indicated agents.
  • Each point represents the mean ⁇ SEM for the group at each time point; n>12 wells for each condition.
  • Dexpramipexole unlike CSA, did not inhibit mitochondrial transition in liver mitochondria.
  • FIG. 3 shows that dexpramipexole and CSA decreased conductance of submitochondrial vesicles (SMVs).
  • Fig. 3a shows an example of a patch clamp recording from a brain-derived SMV; holding potential +60 mV, before and after addition of the indicated agents to the bath (CSA is 1 ⁇ ).
  • Fig. 3b shows histograms represent group data (meaniSEM) depicting effects of the indicated compounds on peak conductance of SMV patches. In all cases current was measured from 0 pA and is presented as peak conductance assuming a linear current-voltage relationship.
  • Fig. 3e shows an ATP decreased peak conductance levels in a concentration-dependent manner. Example shows recordings from a single SMV patch, indicating steady-state changes in conductance level at the indicated concentrations of ATP; holding potential +40 mV.
  • the example shows recordings from a single SMV patch, indicating steady-state changes in conductance level at the indicated concentrations of dexpramipexole; holding potential +50 mV.
  • dexpramipexole inhibits a smaller percentage of peak total current than ATP, which was a general finding in this preparation.
  • One-way ANOVA, pO.0001 pre-selected post hoc Bonferroni corrected t-tests, control vs.
  • Figure 4 shows the dexpramipexole modulation of cellular bioenergetics.
  • Fig. 4b shows dexpramipexole modulation of cellular bioenergetics.
  • Fig. 4b shows dexpramipexole modulation of
  • FIG. 4c shows an example of a single self-referencing oxygen electrode recording from a cultured hippocampal neuron after addition of dexpramipexole (10 ⁇ ) or an equivalent volume of water to the bath.
  • Fig. 4e shows dexpramipexole treatment decreased OCR following both succinate and ADP injection. Primary rat cortical cultures were incubated with digitonin (10 ⁇ g/ml) and rotenone (100 nM) for 45 minutes in MAS1 buffer prior to measurement of oxygen consumption rate (OCR) by a Seahorse ® flux analyzer.
  • OCR oxygen consumption rate
  • FIG. 5 shows that dexpramipexole altered respiration parameters and ATP production in the C2C12 myoblast cell line.
  • FiglO. b. shows that cells compensate for PSI-induced stress, and ATP levels and viability are maintained over the experimental time course. C212 cells were grown for 24 hr.
  • OCR Oxygen consumption rate
  • ECAR extracellular acidification rate
  • Figure 6 shows modulation of complex V activity by dexpramipexole (panels a. and b.).
  • Fig. 6c shows urea-treatment of SMVs alters enzymatic activity, pharmacology of membrane currents and radiolabeled dexpramipexole binding (panels c. ).
  • Fig. 6c also shows c.
  • Fig. 6d shows an immunoblot with an antibody against the Fl ⁇ subunit in protein from control SMVs or urea-treated SMVs.
  • Bottom band shows immunoblot with an antibody against adenine nucleotide transporter (ANT) to provide a loading control.
  • Fig. 6e shows an immunoblot with an antibody against adenine nucleotide transporter (ANT) to provide a loading control.
  • Fig. 6g-i shows binding of radiolabeled dexpramipexole to individual heterologously-expressed subunits of complex V and competition by unlabeled dexpramipexole.
  • Fig. 6g. shows Myc-Flag tagged constructs for human FIFO ATP synthase subunits (as labeled at bottom of gel) immunoprecipitated with anti-FLAG affinity gel and immunoblotted with anti- Myc tag antibody.
  • CTL lane representsimmunoprecipitate with anti-FLAG affinity gel of cell lysate from non-transfected cells.
  • Fig. 6h shows binding of radiolabeled dexpramipexole to individual heterologously-expressed subunits of complex V and competition by unlabeled dexpramipexole.
  • Fig. 6g. shows Myc-Flag tagged constructs for human FIFO ATP synthase subunits (as labeled at bottom of gel)
  • Fig. 6i shows counts per minute of anti-Flag affinity gel immunoprecipitates from cells exposed to 200 nM 14C-labeled dexpramipexole and 20 micro M unlabeled dexpramipexole.
  • Example 2 Methods [00237] Isolation of Brain-derived Mitochondria. Standard techniques were adapted for isolating brain-derived mitochondria. Mitochondria were stored in isolation buffer (IB) at -80 °C.
  • IB isolation buffer
  • PSI-mitochondria male Sprague-Dawley rats (6 wo, ⁇ 200 gm, 3-6 rats/group/condition, at least 2 different preparations/experimental paradigm) were injected with the ubiquitin proteasome inhibitor Z-lle-Glu(OtBu)-Ala-Leu-al (PSI; Peptides International Inc, entucky,USA; s.c. every other day for 1 or 2 weeks; 6.0mg/kg PSI in DMSO, or with DMSO, 200 ⁇ ), and mitochondria isolated and stored as above.
  • SMV submitochondrial vesicle
  • Fl removal from SMVs Fl subunits were removed from SMVs by adapting previously-established methods. 60mg SMV's/lmL IB was treated with ImL of 6mM urea for 5 min. on ice, then centrifuged at 21000xg for lOmin. The pellet was washed 3X in IB (centrifugation at 21000xg for lOmin) and stored in IB.
  • the cells were lysed and the fusion proteins were bound to the EZviewTM Red ANTI-FLAG® M2 Affinity Gel (Sigma, USA), according to the manufacturer's protocol.
  • the proteins were eluted from a portion of the samples and presence of the proteins on the beads was verified by immunoblot analysis, using the mouse anti-Myc antibodyies (Cell signaling Technology).
  • the protein-bound beads were incubated in presence of i 4 C dexpramipexole overnight at 4°C in an end-over-end agitator. They were spun at 3000xg in 0.45um Spin-X centrifugal devices (Corning Life Sciences, USA) for lOmin.
  • Oxygen flux measurements Oxygen uptake was measured in single hippocampal neurons in culture (DIV 14-17). A 2-4 ⁇ (tip diameter) oxygen- sensing electrode oscillated 10 ⁇ close-to and away- from the cell every 3 sec. The difference in current detected at the two positions was translated into oxygen flux. Dexpramipexole (10 ⁇ ) or control solution was added after a 5 min. baseline measurement, and flux was measured for >5 min. post-treatment.
  • Patch-clamp recordings were made from de-energized mitochondria in intracellular solution (120mM potassium chloride, 8mM NaCl, 0.5mM EGTA, lOmM HEPES, pH adjusted to 7.3) at room temperature (22-25°C). Pipettes (80-100 ⁇ ) were filled with the same solution; recordings were made using a Heka 8 amplifier with V m held at positive voltages up to + 180 mV. Data were recorded at 20 kHz and filtered at 500-1000 Hz. Dexpramipexole ( nopp Neurosciences, Pittsburgh, PA) was prepared as a 10 mM aqueous stock and diluted in intracellular recording solution; cyclosporine A (CSA; Sigma, St.
  • CSA cyclosporine A
  • Peak membrane conductance was measured as the peak amount of current (pA) from zero, converted to pS by assuming a linear I-V relationship.
  • Discrete channel conductances were sorted by activity (% occurrence of a conductance level per unit time measured using pCLAMP 10 software, Molecular Devices, Sunnyvale, CA); levels defined as closed (no current), small ( ⁇ 200pS), intermediate (>200pS and ⁇ 750pS) and large (> 750pS).
  • ATP hydrolysis in SMVs was measured with the Bio Vision Aposensor ATP Assay Kit in a 96-well plate with a plate reader (VICTOR3 Multilabel Perkin Elmer). SMVs were suspended in IB plus BSA (0.03 mg/mL), ATP solution (final 0.5mM) and 1 ⁇ of reconstituted ATP-monitoring enzyme. To initiate, 100 ⁇ of Nucleotide Releasing Agent containing Triton X was added and luminescence measured and displayed as percent change in luminescence over time. 3 wells were used for each condition, repeated at least 3X on different SMV isolations.
  • ATP hydrolysis was also measured using an NADH-ATP-synthase kit (Mitosciences, USA; catalog # MS541), according to the manufacturer's protocol with modifications. SMVs were added 20 min. prior to addition of the reagent mix. The rate of change in fluorescence over time as NADH was oxidized was measured as a decrease in absorbance at 340nm.
  • Undifferentiated SH-SY5Y human neuroblastoma cells (ATCC, Manasses, VA) were maintained in humidified 5% C0 2 in 1 :1 Ham's F12 nutrient mixture with Glutamax ® and Minimal Essential Medium with L-glutamine (MEM- Alpha), 10% FBS, and 1% penicillin/streptomycin (Gibco ® Invitrogen Corp., Carlsbad, CA).
  • the C2C12 mouse myoblast cell line (ATCC, Manasses, VA) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco ® ; Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 2mM GlutaMax ® and penicillin/streptomycin. Cells were maintained in 10% C0 2 humidified at 37°C.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • 2mM GlutaMax ® penicillin/streptomycin
  • Cell Viability and Cellular ATP quantitation were measured using CellTiter-Blue ® (Promega Corp., Madison, WI) in black-walled optical imaging multiwell plates, and cellular ATP was measured using the CellTiter-Glo ® (Promega Corp., Madison, WI) luminescence assay in opaque-walled multiwell plates according to the manufacturer's protocol.
  • the assay had 2 modifications: 1) MAS 1 buffer (70mM sucrose, 220mM mannitol, 5mM KH 2 P0 4 , 5mM MgCl 2 , 2mM HEPES, ImM EGTA, 0.2% FA-free BSA; pH 7.2; Sigma- Aldrich, St. Louis, MO) was used instead of Assay Media, and 2) l Ox concentrated digitonin (10 ⁇ g/mL), rotenone ( ⁇ ⁇ ) and dexpramipexole (30 ⁇ ) or control (equivalent volume of water) were added to the cells 45 minutes prior to assay.
  • MAS 1 buffer 70mM sucrose, 220mM mannitol, 5mM KH 2 P0 4 , 5mM MgCl 2 , 2mM HEPES, ImM EGTA, 0.2% FA-free BSA; pH 7.2; Sigma- Aldrich, St. Louis, MO
  • l Ox concentrated digitonin 10 ⁇ g/mL

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Abstract

La présente invention concerne, en partie, des procédés d'identification de composés qui peuvent se lier à un complexe d'ATP synthase, qui augmentent l'efficacité bioénergétique, qui diminuent la consommation d'oxygène ou le taux de consommation de celui-ci, qui augmentent l'efficacité d'utilisation de l'oxygène, ainsi que la survie cellulaire ou toute combinaison de ces effets, et des procédés d'utilisation des composés et/ou des composés identifiés afin d'augmenter l'efficacité bioénergétique, d'augmenter l'efficacité d'utilisation de l'oxygène, de diminuer la consommation d'oxygène, d'augmenter la survie cellulaire ou toute combinaison de ces effets.
PCT/US2011/038159 2010-05-26 2011-05-26 Composés et procédés de modulation de l'efficacité bioénergétique mitochondriale par interaction avec l'atp synthase (complexe v) et ses sous-unités WO2011150221A2 (fr)

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US10456381B2 (en) 2013-08-13 2019-10-29 Knopp Biosciences Llc Compositions and methods for treating plasma cell disorders and B-cell prolymphocytic disorders
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